Dr Robert J R Elliott
email: r.j.elliott@bham.ac.uk

Supreeya Virakul

Multi-Product Firms and Exporting: A Developing Country Perspective

Keywords: .F10, F14, L11 Multinationals, Firm Characteristics, Multi-product, Exports

In this paper we make the distinction between single-product and multi-product firms to contribute to our understanding of the complex relationship between multinational enterprises (MNEs), exporting and economic development. Using firm-level data for Thailand we show that the number of goods produced causes a larger variation in exports volumes than production volumes. Whilst the number of products and the total volume of exports are positively correlated we find, in contrast to US studies, a negative correlation between the number of products produced and the volume of production per product. We then investigate the characteristics associated with multi-product firms and find a distinction between foreignowned and domestic firms. The presence of foreign firms producing single products solely for the domestic market as well as those producing many products for export demonstrates the diversity of behaviour of foreign-owned firms in developing countries.

Introduction

The study of international trade has been transformed by the modelling of firm heterogeneity, productivity and exporting (see e.g. [START_REF] Melitz | The Impact of Trade on Intra-Industry Reallocations and Aggregate Industry Productivity[END_REF][START_REF] Yeaple | A Simple Model of Firm Heterogeneity, International Trade, and Wages[END_REF][START_REF] Melitz | Market Size, Trade and Productivity[END_REF]and Bernard et al. 2007a). What the early literature failed to take into account was that world production and trade is dominated by multi-product firms which has led to recent developments in both the theoretical and empirical literature [START_REF] Eckel | Multi-Product Firms and Flexible Manufacturing in the Global Economy[END_REF][START_REF] Iacovone | Multi-Product Exporters: Diversification and Micro-Level Dynamics[END_REF], Bernard et al. 2007b, Bernard et al. 2006a[START_REF] Nocke | Globalization and Endogenous Firm Scope[END_REF].

The importance of multi-product firms was first revealed for the US by [START_REF] Bernard | Importers, Exporters, and Multinationals: A Portrait of Firms in the U.S. that Trade Goods[END_REF]2006a) who show that 41 percent of firms produce more than one product but that multiproduct firms account for 91 percent of total output while multi-product exporters account for more than 95 percent of total exports. An important element of firm heterogeneity therefore is how firms expand or contract their product range in response to changes in trading conditions.

However, detailed investigations of the multi-product firm phenomenon are limited and almost exclusively concentrated on developed countries. Yet, the role of foreign firms in developing countries is considered a crucial part of the development story with developing countries becoming increasingly aggressive in their approach to attracting foreign direct investment (FDI). Thus, gaining an understanding of the dynamics of introducing new products at the firm level and how government policy can influence the export structure of firms is of direct policy relevance.

In this paper, we examine the role of multi-product firms in a developing country, in this case Thailand. A first pass of the data suggests that there are both similarities and dissimilarities with the US. For Thailand, 43 percent of firms produce more than one product (compared to the 41 percent figure for the US). However, 57 percent of output is produced by multi-product firms and 52 percent of total exports are from firms that export multiple products (compared to the US figures of 91 and 95 percent respectively). The headline figures for the production and exporting share are clearly of a different magnitude to those of Bernard et al. (2006a) for the US. The smaller output percentage for Thailand hints at the differences in the behaviour of firms in developed and developing countries certainly in terms of the size distribution of firms. 1 In this paper we argue that for the case of a newly industrialising country such as Thailand it is important to make the distinction between single and multi-product firms for the following reasons. First, FDI subsidies and tax breaks for foreign firms are often justified by their ability to attract firms and to subsequently benefit from technology and knowledge spillovers. From a spillover perspective, multi-product firms are likely to be more attractive as logically, the greater the number of products produced, the wider the range of technologies employed and thus the greater the likelihood that domestic firms will benefit. The process of a firm becoming multi-product is also associated with process and product R&D which is also strongly associated with positive spillovers.

Second, since the growth through exporting route has proved to be particularly successful for many East Asian countries over the last two decades, governments are likely to prefer investment from foreign firms that produce more than one product as this will increase the likelihood of exporting at least one of the products. Third, multi-product firms may be more attractive to host governments as they should exhibit less susceptibility to demand shocks as 1 Comparisons between our results and [START_REF] Bernard | Importers, Exporters, and Multinationals: A Portrait of Firms in the U.S. that Trade Goods[END_REF]Bernard et al. ( , 2006a) must be made carefully as the d efinition of whether a firm is multiple product or not depends on how a product is defined. The greater the level of disaggregation, the larger the number of multi-product firms. In this paper we define a p roduct acco rding to the equivalent of the 5-digit Intern ational Standard Industrial Classification (ISIC). Bern ard et al. (2006a) use a 5-digit US SIC classification and Bern ard et al. (2005) use a 10-digit Harmonised System (HS) classification to measure their output and export statistics respectively and is probably one explanation for at least some of the differen ce in our headline figures. The SIC 5-digit data consists of around 1,800 products whilst the HS 10-digit data contains 8,500 products of which two thirds are from the manufacturing sector. The data reveal that firms produce across four and even 2-digit industries and that the product distribution tends to be highly skewed where for example, exports of one produ ct in a multi-product firm may account for considerably more than 50 p ercent of total exports. See Bernard et al. (2006a) for further discussion. the risk from, for example, changes in fashion or advances in product-specific technology, is spread over a variety of exports and possibly export markets. Hence, domestic employment may benefit from a smoothing effect.

In this paper we provide an analysis of the structure of foreign firms and the characteristics of firms that produce multiple products which should provide a useful insight into the role of MNEs in developing countries. More specifically we examine two aspects of the multiproduct and development question. In the first stage we examine the relationship between multi-product firms' extensive margins (number of products produced or exported) and intensive margins (output or export sales per product). Given that changes in trade barriers or trade costs will lead to intra-firm adjustment along firms' extensive and intensive margins we examine how this relationship affects the distribution in firm size. In addition, we examine the correlation between firms' extensive and intensive margins.

In the second stage of the paper, we examine the characteristics associated with multiple product producers making a distinction between domestic and foreign-owned firms. A complex picture of the behaviour of MNEs in developing countries emerges where foreignowned firms that export are strongly associated with being multi-product but foreign firms that only serve the domestic market show a strong negative partial correlation with being multiproduct. These factors might explain, in part, why evidence for knowledge diffusion and productivity spillovers is less widespread that one might expect. Our finding that a significant proportion of foreign-owned firms supply only the domestic market and produce just a single product is an interesting new stylised fact.

The structure of the remainder of this paper is organised as the follows. Section 2 describes the data. In section 3, we discuss our empirical model and present the results of our intensive and extensive margin analysis while section 4 presents our results examining the characteristics of those firms that produce multiple products and the factors related to the number of goods produced. Section 5 concludes.

Descriptives and Data

Thailand has been the third largest exporter from the Southeast Asian region over the last 10 years (ASEAN Statistical Yearbook, 2005). As a member of ASEAN, Thailand shares in the benefits of the ASEAN Free Trade Area. 2 Not surprisingly, the ASEAN region remains a major export market for Thailand. The share of Thai exports to ASEAN in 2007 was about 21.3 percent of total exports with 12.6 percent and 12.8 percent exported to the US and EU-15 respectively. Since 1999, the total export value of trade has increased dramatically reaching US$ 152,477.58 million in 2007. In contrast to many developed countries, the manufacturing sector still dominates, accounting for 78 percent of total exports in 2007.

For Thailand, sectors with a large volume of exports tend to be high-technology products such as computers, parts, and accessories, automobiles and parts, and integrated circuits. The production of computers and parts has been Thailand's leading industrial export sector for many years, accounting for 11.35 percent of the country's total exports in 2007. The second leading export industry is the automotive industry.

Numerous foreign automotive manufacturers from Japan, the US and Europe are based in Thailand and use the country as an export platform to sell their products to the rest of the world. Other prominent export sectors include labour-intensive products such as gems, jewellery, and garments. -2004. 4 The data contain detailed information on standard firm-level variables such as structure of ownership, employment, region, wage, productivity, research and development (R&D), output and exports. One significant advantage of this data is that we are able to identify the number of products a firm produces. Our product classification is based loosely on ISIC classifications of what constitutes a product and are based on the question in the survey that asks the firms to "list the products that you produce". We believe this approximates to a 5-digit product classification.5

When we examine trends at the 2-digit ISIC level for the four years of our sample 2001 to 2004 we observe that the sectors that have a high percentage of exporting firms of more than 70 percent are ISIC 18 (Wearing Apparel; dressing and dying of fur), ISIC 32 (Radio, television and communication equipment) and ISIC 36 (Furniture). In 17 out of 22 2-digit ISIC sectors we observe an increase in the proportion of firms that export with ISIC 34 (Motor vehicles, trailers & semi-trailers) showing the largest increase in exports during this period.

In Table 1 we present the share of output and the share of firms that produce single and multiple products across various groupings. When we consider all firms, we see that the majority of firms produce only one product (57.12 percent) with 17.81 percent producing two products and only 9.15 percent producing five or more products.6 However, those 57.12 percent of firms only produce around 43 percent of total output with the 9.15 percent of firms producing five or more products producing 15 percent of total output. If we compare foreignowned and domestic firms we observe that a larger proportion of domestic firms produce just one product. Thus, consistent with [START_REF] Bernard | Multi-Product Firms and Trade Liberalization[END_REF] we find that foreign firms have a higher likelihood of being multi-product and a higher share of output with 17.25 percent of firms producing five or more products. Comparing exporters and non-exporters is also illuminating where we find an even greater difference with 61.16 percent of non-exporters and only 53.15 percent of exporters producing a single product.

[Table 1 about here]

Finally, we introduce a final complication by making a distinction between foreign-owned exporters and non-exporters. We find that 68 percent of foreign non-exporters produce a single product. The fact that approximately one fifth of foreign firms do not export is a stylised fact that we believe has not been previously highlighted in the literature and suggests that FDI may be substituting for exports for a significant number of firms even in the context of a relatively small developing country. This insight adds a layer of complexity to our analysis and hints at a more subtle relationship between foreign firms and the benefits accrued to the host country.

Multi-Product Firms' Intensive and Extensive Margins

As previously noted, multi-product firms in Thailand produce 57 percent of total output while firms that export multiple products account for over 52 percent of total export sales. [START_REF] Bernard | Multi-Product Firms and Trade Liberalization[END_REF] investigate this phenomenon for multi-product firms in the US by examining the contribution of firms' extensive margins to firm-size distribution. Similarly, [START_REF] Yeaple | A Simple Model of Firm Heterogeneity, International Trade, and Wages[END_REF] argues that large firms are responsible for much in the variation in sales across firms managing product lines much more actively than small firms. This line of thinking is matched by [START_REF] Berger | Diversification Effect on Firm Value[END_REF] who find single product firms have larger sales per product than multiproduct firms. In this section we follow [START_REF] Bernard | Multi-Product Firms and Trade Liberalization[END_REF] to examine the relationship between intensive and extensive margins and size distribution for Thailand where the importance of attracting large MNEs is often part of government industrial policy. [START_REF] Bernard | Multi-Product Firms and Trade Liberalization[END_REF] begins with a cross-section estimation. The basic framework for firmsize distribution is to identify firm' extensive (number of products) and intensive (output per product) margins. We have a panel estimation so the relationship is presented in equation ( 1),

it it it Y n y (1)
where i Y is firm size measured by total output of each individual firm, i n is the number of products produced by firm and i y is the average output per product that is defined as

1 it pit p it yy n .
The subscripts i , t and p denote firm, time and product respectively. The relationship between firm size and multiple product firms requires a knowledge of how firm size varies. By taking the log of equation ( 1), the model can be separated into two regressions for firms' intensive and extensive margins as a function of the log of total output, 1. Thus the coefficient of 1 captures the partial correlation between total output and the extensive margin and 2 captures the partial correlation between total output and the intensive margin [START_REF] Bernard | Multi-Product Firms and Trade Liberalization[END_REF]).

In addition, we examine the relationship between exporting and firms' intensive and extensive margins. In the case of an exporting firm, total exports is the number of products exported (5)

Since a firm's extensive and intensive margins are related by construction through an accounting identity (the log of the two margins sums to the log of total exports), where 21 1 and 43 1 we simply report the estimated results of a firm's extensive margin ( 1 and 3 ). A robust variance estimation corrects for the problem of heteroscedastic errors. The results from OLS estimations with and without region, industry and time fixed effects are presented in Table 2 and are based on a sample of multi-product firms only.

In Columns (1) and ( 2), we find that the number of products produced accounts for approximately one percent of the variation in total firm output. This means that an increase in the number of products (extensive margin) accounts for only one percent of the increase in total output. On the other hand, this result indicates that the variation of total firm output in Thailand is mainly due to changes in average output per product (intensive margin).7

A slightly higher variation is observed if we consider the number of products exported and total export sales (Columns 3 and4). The coefficient shows that the number of products exported causes a variation in total export sales of 7.4 percent. This means that the number of products exported raises total export sales by 7.4 percent by keeping average export sales per product constant.

[Table 2

)
In Table 3 we observe a positive correlation between the extensive and intensive margins in Columns (3) and (4) only. Whether the correlation is positive, negative or insignificant depends on the functional form of the distribution of shipments across products within firms.

In this case, the positive relationship indicates that the number of products exported increases export sales per product by between 50.1 percent and 58.4 percent. We can conclude therefore that multi-product firms only marginally increase the number of products exported but for each product, multi-product firms export a larger volume of each. However, in contrast to [START_REF] Bernard | Multi-Product Firms and Trade Liberalization[END_REF] and [START_REF] Iacovone | Multi-Product Exporters: Diversification and Micro-Level Dynamics[END_REF], we find a negative and significant correlation for firms' extensive and intensive margins when we consider production data. Thus, in Columns ( 1) and ( 2), we find that an increase in the number of products produced decreases the amount of output per product by between 64.1 percent and 69.2 percent. This negative correlation is consistent with the relationship predicted by the models of [START_REF] Nocke | Globalization and Endogenous Firm Scope[END_REF], and [START_REF] Eckel | Multi-Product Firms and Flexible Manufacturing in the Global Economy[END_REF]. The empirical result suggests that in Thailand, the more products a firm develops, the less of each one is produced. This can be explained by diseconomies of scope in the production unit of multi-product firms in Thailand and inefficiency in monitoring various production process.8 Another explanation is that there may be advantages associated with the production of a number of products and that by using the same production unit, distributing products through the same channels and managing production within the same organisation there is no discernible difference in cost. A third explanation is that multi-product firms in Thailand may be trying to expand their market potential by increasing the number of products produced rather than merely increasing sales of existing products. If firms produce a greater number of products it may help to reduce future risk resulting from the product life cycle at any given period.

[Table 3 about here]

From the decomposition of the firm-size distribution and firms' extensive margins, we found that intra-firm adjustment on the number of products produced and exported by multiproduct firms positively and significantly affects the variation in firm size (the classification for production and export data is the same). The effect on the variation in firm size is mainly due to changes in output and export sales per product. When we consider the relationship between firms' extensive and intensive margins, our results show that extensive and intensive margins are negatively correlated in production but positively correlated in exporting.

We now know that multi-product firms also play a significant but complex role in Thailand's economy. Although there are a larger number of single product firms, approximately 57 percent of total output is accounted for by multi-product firms. Given the importance of multi-product firms we now investigate which factors, in addition to size, are associated with a firm's decision to produce multiple products.

The Characteristics of Multi-Product Firms

Being a Multi-Product Firm

Recent stylised facts have shown that, in both domestic and international markets, multiproduct firms have become increasingly important. We now investigate the characteristics of those firm's that produce multiple products.

We estimate a pooled probit model for the binary dependent variable, which indicates the status of a firm.9 All independent variables are lagged by one year in order to mitigate against possible simultaneity problems. Unfortunately the data does not provide a set of instruments to control for possible exogeneity between multi-product production and our dependent variables. For example, being multi-product may cause total factor productivity (TFP) to rise or make it more likely that a firm will export. We believe this is less of a problem than with the traditional determinants of exporting regressions. However, we acknowledge that lagging by one year is not ideal and hence in our results section we refer to associations and partial correlations instead of determinants and effects. Thus, our probit model is as follows,

( 1) ( 1)

Pr( 1 ) ( )

it i t i t MULTIDUM Z Z (8)
where, it MULTIDUM is a dummy variable that is 1 if the firm is multi-product and 0 otherwise. The term Z is a vector of firm characteristics and is the cumulative distribution function of the normal distribution function.

We include five region dummies, twenty-three 2-digit industry and two year-dummies in order to control for unobserved effects. 10 In addition, we allow for robust clustering at the 2-digit industry level (clustering at the regional level made little difference to the results). This relaxes the independence assumption and requires only that the observations are independent across sectors. In equation (8), the vector of firm characteristics Z includes the following:

EX is an export dummy which equals 1 if the firm has positive export sales and 0 otherwise.

FOREIGN is a dummy, which equals 1 if at least 10% of shares are foreign owned, and 0 otherwise. Cut-offs of 25% and 50% were used in a sensitivity analysis.

EX*FOREIGN is an interaction term that measures the effect of being both foreign and an exporter over and above the individual effects.

LP TFP is a measure of total factor productivity. The calculation of the variable is obtained from the semi-parametric approach of [START_REF] Levinsohn | Estimation Production Functions Using Inputs to Control for Unobservables[END_REF] which takes account of unobserved firm-specific productivity shocks. In a sensitivity analysis, we use two alternative measures of TFP. The R&D estimator of TFP () BUETTNER TFP is obtained from a semi-parametric and nonlinear least square regression of [START_REF] Buettner | R&D and the Dynamics of Productivity[END_REF] that allows for endogenous R&D.

The standard labour productivity () LABPRO D TFP is calculated from the log of value added over total labour.11 size is measured as the log of total employment. As a robustness check we also categorise firm size into small () SMALL , medium () MEDIUM , large () LARGE and very large () VLARGE by following the quartile distribution of the total employment for all firms operating in the same 2-digit ISIC (Rev.3).

w age is the log of wage per employee. Wage is an indicator of labour quality. It is expected that the higher the wages, the more superior the quality of labour and the more likely that a firm will be able to produce multiple products.

RDPRODUCT and RDPROCESS are dummy variables for R&D to capture those firms that undertake R&D in product development and production processes respectively. R&D activity is an important mechanism for firms to introduce new products [START_REF] Brander | Product Line Rivalry[END_REF]. R&D is also an important procedure for enhancing the quality of existing products and for developing new products as well as highlighting cost savings. It is expected that a firm that carries out R&D especially product R&D is more likely to be multi-product.

The results reported in Tables 4 and5 are marginal effect estimations that are calculated at the mean of the independent variables except for dummy variables. Each coefficient indicates the change in the probability of the outcome. Our variables are defined and descriptive statistics presented in Tables 8 and9 in the appendix respectively. It should be noted that the results are based on reduced form regressions for Thailand data so they cannot be generalised to other contexts.

In Table 4, the results of our preferred specification in Columns (3) and ( 4) show a complex relationship between export status and the probability of a firm to be a multi-product producer. The results suggest that for Thailand it is not whether you are an exporter that is important but the export status of the firm combined with our ownership variable. For example, being foreign and an exporter has a large positive partial correlation with being a multi-product producer. In contrast, being an exporter per se is insignificant. This suggests a difference in behaviour between domestic and foreign exporters.

In Thailand, foreign ownership appears therefore to have an important association with multiproduct production although it is not a straightforward relationship. The individual partial correlation for foreign ownership is negative and significant for all specifications. This suggests that foreign-owned firms per se are negatively associated with multi-product production. This is a surprising result. One explanation might be overseas firms setting up single product assembly plants that specialise in the production of one single product for sale either domestically in Thailand or for export (possibly to Thailand's ASEAN neighbours).

This would also fit with the [START_REF] Baldwin | Multiproduct Multinationals and Reciprocal FDI Dumping[END_REF] hypothesis that MNEs locate the production of different varieties in different countries. However, as noted earlier, foreignowned firms that also export are positively and significantly correlated with firms that produce multiple products. Thus it is clear that foreign firms cannot be considered one homogenous group. A further possible explanation is that foreign-owned firms which export and foreignowned firms which serve the domestic market maybe engaged in either horizontal or vertical FDI. 12For TFP, as expected we observe that highly productive firms are positively associated with multi-product firms. The positive and significant coefficients for product R&D and process R&D suggests that firms that carry out R&D in either product development or production processes, or both, are positively related to the probability that a firm will be a multi-product producer. When we examine our proxy for the quality of labour we see that the coefficient on wage is positive but generally insignificant.

As expected, the relationship between size and being a multi-product firm is positive and significant at the one percent level. Increasing firm size by one unit is associated with an increase in the probability of producing multiple products of approximately 6 percentage points. If we categorise firm size into small, large and very large firms, the coefficient s are also significant at the one percent level with small firms being negatively correlated with being multi-product producers. As firm sizes increases, we observe increasingly positive results so that the larger the size, the greater the probability of producing multiple products.

To further investigate the negative foreign ownership and exporter results from Table 4 we split the sample into domestic firms and foreign firms. Approximately one quarter of our firm sample are foreign-owned firms. We retain the 10 percent foreign-owned definition. 13 The results are presented in Table 5. The insignificant coefficient for export status in Table 4 is now explained. Observe that the export status of Thai domestic firms has no relationship with the probability of a firm producing multiple products. In contrast exporting has a significant and positive partial correlation with the probability of a foreign firm being a multiproduct producer and is picked up in Table 4 by the positive and significant interaction term.

This suggests a systemic difference between the behaviour of foreign and domestic firms with foreign exporters producing more than one product and domestic exporters tending to concentrate on the export of a single product. The larger number of domestic firms explains why the overall figure in Table 4 is insignificant (6,878 domestic against 2,643 foreign firms). 13 In a sensitivity analysis we tested 25 percent and 50 percent cut-off points with broadly similar results.

For productivity, the coefficients for both domestic and foreign firms are positive and significant for only four of our twelve specifications. For process R&D, the positive significant coefficients for the domestic sample indicate that for domestic firms, R&D in production processes is associated with a higher probability of a firm becoming multi-product producer. In contrast, the insignificant coefficient for our foreign firm sample suggests that neither R&D process development nor wages are associated with an increase in the probability of being a multi-product producer. However, R&D product development is positive and significant at the one percent level for domestic firms and five percent for foreign firms except in Column ( 9). Firm size for both domestic and foreign firms is positive and significant.

Our results suggest therefore that for Thailand the relationship between ownership and multiple product production is complex. We observe that individually foreign-owned firms and exporters have a negative partial correlation with the likelihood of being a multi-product producer but that being foreign and an exporter means a firm has a positive partial correlation with the production of multiple products.

[Table 4 and 5 about here]

The Number of Products Produced

In the previous section we examined the characteristics of being a multi-product firm. In this section we identify a firm's performance by investigating the characteristics associated with the number of products produced. Thus, our dependent variable is now a count of the number of products produced. A simple histogram of the distribution of the number of products reveals that approximately 50% produce just one product, 20% two products and 10% for three, four and five products with only 1% producing six or more.

Since count data is used as our dependent variable, there are two alternative regression models for counts which are poisson regression model and negative binomial regression model. 14 In this paper, we estimate count data using a negative binomial regression model. Additionally, we also estimated a simple poisson count model for a sensitivity check. 15 We lag all independent variables by one year to avoid possible simultaneity problems. As this is not ideal we continue to avoid direct causal language in discussing our results. Our negative binomial regression model can be specified as follows,

11

1 1 1 () Pr( ) ! ( ) NPRO DUCT NPRO DUCT NPRO DUCT Z NPRO DUCT (9)
where NPRODUCT is a count for the number of products produced by each firm.

Z is a vector of firm level characteristics. () is the gamma function. is the degree of overdispersion which equals to zero when negative binomial and poisson has the same distribution. Finally, is known as the observed heterogeneity and is estimated from the observed firm characteristic where exp( ) Z

. 16 In equation (9), the independent variables included in a vector of firm-level characteristics () Z are the same as before. Five region, 2-digit ISIC industry and two year-dummies are included in order to control for unobserved effects. A robust variance estimation corrects for possible heteroscedasticity in the error term and we allow for clustering at the 2-digit industry level.

Tables 6 and7 present the coefficients obtained from the estimation of marginal effects for 14 Poisson regression estimation assumes that the observed count is drawn from a poisson distribution of which the mean and varian ce are equal. In practice, the poisson regression model may be inappropriate due to overdispersion. Therefore, the negative binomial regression model which is an extension of Poisson regression alleviates th e over dispersion problem by in cluding a parameter that captures unobserved heterogen eity. 15 The estimated results from poisson regression are identical to the negative binomial regression. This indicates that we do not have a problem with over dispersion in our data. 16 According to [START_REF] Long | Regression Models for Categorical and Limited Dependent Variables[END_REF] and [START_REF] Cameron | Regression Analysis of Count Data[END_REF], exp( ) is unknown but it can be drawn from a gamma distribution of which mean equals 1 and varian ce equals .

our negative binomial regressions calculated at the mean of the independent variables except for the dummy variables.

In general, the sign and significant level of results in Tables 6 and7 are consistent with those presented in Tables 4 and5. Table 6 shows that being an exporter does not have any significant association with the number of products produced. For ownership status, the relationship of foreign ownership and the product count is not so simple. The negative and significant coefficient indicates that being a foreign-owned firm is negatively associated with the number of products produced. However, the interaction term between being a foreignowned firm and an exporter has a positive effect.

TFP has a significant positive impact on the number of products produced in two of the six columns. For example, the TFP coefficient in Column (1) indicates that increasing TFP by one unit is associated with 10.8 percentage points increase in the expected change in the product count. Other variables such as R&D of both product and production process, wage, size have positive and significant effect on the number of products produced as expected.

In Table 7, the sample is split into domestic and foreign firms. The insignificant results for export status in Table 6 are now explained. This is also picked up by the positive and significant results for the interaction term in Table 6. The export status of domestic firms has no significant association with the product count. In contrast, the export status of foreign firms has a positive and significant impact on the number of products produced. For example, the EX coefficient in Column ( 12) indicates that being a foreign exporters is associated with 30.8 percentage points increase in the expected change in the number of product count.

When we consider the productivity of domestic firms, the coefficient is positive and significant when size is excluded. In the foreign firms' sample, the coefficients of TFP are generally positive and significantly associated with the number of products produced. In both samples, product R&D and process R&D have positive coefficients but are only significant in the sample of domestic firms. Wage of only domestically owned firms is associated with an increase in the expected change in the number of products produced. As expected, firm size of both domestic and foreign firms is positive and significant. A one unit change in firm size is associated with a proportional increase in the expected change in the number of products by 16 percentage points for domestic firms and 18 percentage points for foreign firms.

Finally, it is worth pointing out that we performed a series of sensitivity checks. For ownership structure, we tested 25 percent and 50 percent foreign owned as the cut-off point.

For productivity, the [START_REF] Buettner | R&D and the Dynamics of Productivity[END_REF] approach and standard labour productivity were employed instead of our [START_REF] Levinsohn | Estimation Production Functions Using Inputs to Control for Unobservables[END_REF] approach. 17 The results are broadly consistent with results shown in Tables 4 to 7 but are not included for reasons of space.

[Tables 6 and7 about here]

Conclusions

In this paper, we investigate different aspects of multi-product firms in international trade using the Annual Survey of Thailand's manufacturing industry from 2001 to 2004. The empirical analysis comprises two sections. First, we examine the relationship between multiproduct firms' extensive margin (number of products) on output or exporting. Second, we investigate the characteristics associated with being a multi-product firm using binary data and the number of products produced using count data. The use of the former allowed us to analyse the characteristics of those multi-product firms while the latter is used to explain factors that affect the number of products produced. We also examine the systematic differences between domestic and foreign firms by estimating each sample separately.

Our results show that little variation is observed for firms' extensive margins in both total output and export sales. However, firms' extensive margins seem to have a higher variation in export sales than in total output. We suspect a partial explanation for these low variations, at least relative to the findings in [START_REF] Bernard | Multi-Product Firms and Trade Liberalization[END_REF], is because of the level of aggregation we use when we classify the number of products. Another explanation arises from the fact that multi-product firms in Thailand do not dominate domestic production and exporting.

Various factors such as export status, foreign ownership, TFP, R&D both in product and in the production processes and firm size are important correlates with both multi-product firms and the number of products produced. Productive and large firms and those that carry out R&D also have a strong association with being a multiple product firm. Similarly, the effects of different factors on the expected number of products produced by firms are generally consistent with the factors associated with the probability of becoming a multi-product firm.

We did however find that there are systematic differences in the factors correlated with multiproduct production between different groups in our sample of Thai firms. The differences in the significance and sign of factors indicate that domestic firms perform differently to foreign firms. Although our results for Thailand cannot be generalised, from a development policy perspective the weak association between R&D and the propensity of a foreign firm to be multi-product or the number of products produced is of interest. Assuming that potential benefits from spillovers increase with the number of varieties this may partia lly explain the lack of evidence for spillovers found in many studies. In contrast, it could be argued that a technologically advanced single-product firm could offer greater potential spillovers than a less technologically advanced multi-product firm.

In sum, for Thailand we show therefore that the relationship between MNEs and development is complex. We show that multi-products firms have played a significant role in international trade especially though exporting and FDI. The results from the empirical analysis also confirm that being foreign owned and an exporter is an important characteristics associated with the emergence of multi-product firms and number of products produced. There appears however to be differences in the behaviour of foreign firms in developing and developed countries. In future research it would be useful to break down foreign ownership into country of origin to see whether there is a difference between the behaviour of firms from developing and developed countries. A further extension that would require a longer time period would be to examine the behaviour of firms in response to a shock to see whether product adjustment occurs at the intensive or extensive margin. of production processes and 0 otherwise.

BK K M

A dummy variable identifies whether firm locates in Bangkok and Metropolitan Area or not.

CENTRAL

A dummy variable equals 1 if a firm locates in Central region excluding Bangkok and Metropolitan Area and 0 otherwise.

EAST

A dummy variable equals 1 if a firm locates in Eastern region and 0 otherwise.

NORTH

A dummy variable equals 1 if a firm locates in the North of Thailand and 0 otherwise.

SOUTH

A dummy variable equals 1 if a firm locates in the South of Thailand and 0 otherwise.

  and by using OLS estimation techniques, 12

Table 1 : Share of Firms and Output for Different Groups by Product Distributions

 1

	Number products produced	All Firms Share Share Firms Output	Domestic Firms Share Share Firms Output	Foreign Firms Share Share of Firms Output	Non-Exporting Firms Share Share Firms Output	Exporting Firms Share Share of Firms Output	Foreign Non-Exporting Firms Share Share of Firms Output	Foreign Exporting Firms Share Share of Firms Output
	1	57.12	43.02	58.17	42.49	54.37	43.31	61.16	52.63	53.15	40.54	68.29	48.34	50.85	42.75
		(5,438)		(4,001)		(1,437)		(2,883)		(2,555)		(364)		(1,073)
	2	17.81	19.79	16.89	20.19	20.20	19.58	16.31	20.79	19.28	19.57	16.70	22.76	21.09	19.21
		(1,696)		(1,162)		(534)		(769)		(927)		(89)		(445)
	3	9.16	13.74	9.57	16.91	8.10	11.42	8.59	17.14	9.71	12.95	6.38	21.99	8.53	10.25
		(872)		(658)		(241)		(405)		(467)		(34)		(180)
	4	6.76	8.66	6.54	8.87	7.34	8.44	5.11	4.59	8.38	9.60	3.75	3.51	8.25	9.01
		(644)		(450)		(194)		(241)		(403)		(20)		(174)
	5+	9.15	14.79	8.83	11.54	9.99	17.25	8.82	4.85	9.47	17.33	4.88	3.41	11.28	18.78
		(871)		(607)		(264)		(416)		(455)		(26)		(238)
	Total	100	100	100	100	100	100	100	100	100	100	100	100	100	100
		(9,521)		(6,878)		(2,643)		(4,714)		(4,807)		(533)		(2,110)
	Note: Numbers of observation are reported in parentheses.

Table 2 : OLS Regression Decomposition of Firm Size and Firms' Extensive Margins

 2

			Production		Exporting
		(1)	(2)	(3)	(4)
	Y ln it	0.009*** (4.17)	0.012*** (5.20)
	ln e Y it			0.074*** (21.87)	0.074*** (20.48)
	Observations	6042	6042	3331	3331
	R-squared	0.003	0.057	0.118	0.189
	Additional	None	Region, Industry	None	Region, Industry
	Covariates		and Time Fixed		and Time Fixed
			Effects		Effects
	Note: Sample in cludes multi-product firms only. Dependent variable in Column (1) and (2) is the log of number of
	products produ ced (ln ) it n , and Column (3) and (4) is the log of number of produ ct exported (ln ) e it n . Robust t -statistics in parentheses. *** significant at 1%.

Table 3 : OLS Regression of Firms' Extensive and Intensive Margins

 3

					Production	Exporting
				(1)	(2)	(3)	(4)
	ln	n	it	-0.692*** (9.32)	-0.641*** (9.22)
	ln e it n			0.584*** (7.49)	0.501*** (6.33)
	Observations	6042	6042	3331	3331
	R-squared	0.014	0.200	0.018	0.139
	Additional	None	Region, Industry	None	Region, Industry
	Covariates		and Time Fixed	and Time Fixed
					Effects	Effects
	Note: Sample in cludes multi-product firms only. Dependent variable in Column (1) and (2) is log of output per
	product (ln ) it y , and Column (3) and (4) is the log of export sales p roduct per produ ct (ln ) e it y . Region, industry and time dummies are in cluded. Robust t-statistics in parentheses. *** significant at 1%.

Table 4 : The Characteristics Associated with Multiple Product Producers (Dep. Var. is

 4 Robust z-statistics in parenth eses. Standard erro rs are adjusted for clustering at the 2-digit industry level. Region, 2-digit industry and time dummies are in cluded. All the dependent variables are lagged one year. * significant at 10%; ** significant at 5%; *** significant at 1%.

	it MULTIDUM )
									(1)	(2)	(3)	(4)	(5)	(6)
	EX	( 1) it					0.031 (1.00)	0.033 (1.04)	-0.029 (0.86)	-0.030 (0.86)	-0.024 (0.69)	-0.023 (0.67)
	FOREIGN	( 1) it		-0.139*** (4.33)	-0.139*** (4.30)	-0.159*** (4.77)	-0.160*** (4.77)	-0.158*** (4.73)	-0.159*** (4.72)
	(	* EX FOREIGN	( 1) it )	0.127*** (2.67)	0.127*** (2.68)	0.144*** (3.20)	0.145*** (3.22)	0.145*** (3.10)	0.146*** (3.12)
				LP					0.056***	0.057***	0.019*	0.019*	0.023**	0.024**
	( 1) it TFP					(4.85)	(4.86)	(1.85)	(1.82)	(2.14)	(2.13)
	RDPRODUCT it ( 1)		0.093*** (5.51)	0.067*** (4.48)	0.076*** (5.18)
	RDPROCESS	( 1) it			0.102*** (4.63)	0.083*** (4.08)	0.088*** (4.02)
	wage	( 1) it					0.000 (0.00)	-0.001 (0.02)	0.023 (1.20)	0.023 (1.19)	0.019 (0.93)	0.019 (0.91)
	size	( 1) it						0.058*** (6.63)	0.059*** (6.68)
	SMALL	( 1) it				-0.078*** (4.73)	-0.078*** (4.73)
	LARGE it ( 1)				0.072*** (3.01)	0.071*** (3.01)
	VLARGE it ( 1)			0.132*** (4.74)	0.133*** (4.75)
	Observations				9,521	9,521	9,521	9,521	9,521	9,521
	Note:

Table 5 : The Characteristics Associated with Multiple Product Producers by Ownership (Dep. Var. is

 5 Robust z-statistics in parentheses. Standard errors are adjusted for clustering at the 2-digit industry level. Region, 2-digit industry and time dummies are in cluded. All the dependent variables are lagged one year. * significant at 10%; ** significant at 5%; *** significant at 1% .

	it MULTIDUM )

Table 6 : The Characteristics Associated with the Number of Products Produced (Dep. Var. is

 6 z-statistics in parentheses. Region, 2-digit industry and time dummies are included. All the dependent variables are lagged one year. * significant at 10%; ** significant at 5%; *** significant at 1%.

				it NPRO DUCT )
									(1)	(2)	(3)	(4)	(5)	(6)
	EX	( 1) it					0.035 (0.39)	0.039 (0.44)	-0.144 (1.57)	-0.144 (1.57)	-0.120 (1.27)	-0.117 (1.25)
	FOREIGN	( 1) it		-0.404*** (6.07)	-0.405*** (5.98)	-0.454*** (6.92)	-0.455*** (6.88)	-0.449*** (6.76)	-0.451*** (6.69)
	(	* EX FOREIGN	( 1) it )	0.411*** (3.44)	0.410*** (3.46)	0.462*** (4.44)	0.463*** (4.50)	0.464*** (4.14)	0.464*** (4.18)
				LP					0.108***	0.109***	-0.001	-0.001	0.018	0.019
	( 1) it TFP					(4.78)	(4.88)	(0.04)	(0.05)	(0.79)	(0.80)
	RDPRODUCT it ( 1)		0.288*** (6.21)	0.202*** (5.50)	0.233*** (5.97)
	RDPROCESS	( 1) it			0.302*** (4.82)	0.238*** (4.22)	0.255*** (4.21)
	wage	( 1) it					0.037 (0.69)	0.035 (0.64)	0.106** (2.34)	0.105** (2.32)	0.088* (1.81)	0.087* (1.77)
	size	( 1) it						0.167*** (7.91)	0.169*** (8.07)
	SMALL	( 1) it				-0.213*** (4.59)	-0.213*** (4.52)
	LARGE it ( 1)				0.210*** (2.83)	0.210*** (2.82)
	VLARGE it ( 1)			0.373*** (5.80)	0.379*** (5.86)
	Observations				9,521	9,521	9,521	9,521	9,521	9,521
	Notes:

Table 7 : The Characteristics Associated with the Number of Products Produced by Ownership structure (Dep. Var. is

 7 z-statistics in p arentheses. Region, 2-digit industry and time dummies are in cluded. All the dependent variables are lagged one year. * significant at 10%; ** significant at 5%; *** significant at 1% .

	it NPRO DUCT )

Table 9 : Descriptive Statistics

 9

	Variable				Obs	Mean	Std. Dev.	Min	Max
	ln	Y	it					6,042	14.81	2.21	6.31	20.61
	ln	y	it					6,042	13.73	2.22	5.21	19.80
	ln	n	it					6,042	1.08	0.38	0.69	2.30
	it ln e Y					3,331	14.70	2.36	3.86	20.37
	it ln e y					3,331	13.87	2.23	3.86	19.21
	it ln e n					3,331	0.83	0.51	0	2.08
	MULTIDUM		9,521	0.43	0.49	0	1
							it
	NPRODUCT		9,521	1.95	1.38	1	10.00
							it
	it ( 1) EX				9,521	0.50	0.50	0	1
	it ( 1) FOREIGN	9,521	0.28	0.45	0	1
	FOREIGN	( 1) it 25	9,521	0.25	0.43	0	1
	FOREIGN	50	( 1) it	9,521	0.14	0.35	0	1
	it ( 1) LP TFP				9,521	9.22	1.84	0.47	16.69
	it ( 1) BUETTNER TFP		9,195	10.19	1.28	1.21	15.31
	it ( 1) LABPRO D TFP				9,521	8.98	1.05	1.45	14.00
	it ( 1) RDPRODUCT	9,521	0.08	0.27	0	1
	it ( 1) RDPROCESS	9,521	0.06	0.24	0	1
	w age	it ( 1)				9,521	7.71	0.53	4.19	10.29
	size	it ( 1)				9,521	4.79	1.50	1.10	9.00
	it ( 1) SMALL				9,521	0.26	0.44	0	1
	it ( 1) LARGE		9,521	0.25	0.43	0	1
	it ( 1) VLARGE		9,521	0.25	0.43	0	1
	,

Each year, some firms do not respond or even shut down which causes our data set to have an unbalan ced structure. To compensate for the closure or none response of some firms, in 2004 the sampling was extended and data collected fo r additional plants(OIE, 2004). Unfo rtunately we do not have sp ecific data on firm deaths.

Produ ct identification match ed produ ct lists with the I SIC 5-digit classification by visual inspection.

Our figures are broadly consistent with a study by[START_REF] Goldberg | Multi-Product Firms and Product Turnover in the Developing World: Evidence from India[END_REF] who find that for manufacturing firms in India the single-p roduct and multi-product firms account for 53 and 47 percent respectively.

As Bernard et al. (2006a) point out, our use of the equivalent of 5-digit ISIC data will have the effect of masking unobserved changes within 5-digit categories thus our results are likely to underestimate the importan ce of firm adjustments to the extensive margins.

Diseconomies of scope o ccu rs when a firm faces higher marginal costs of produ ction if the new production lines are added and therefore causes a reduction of the existing produ ct line.

Sin ce our data has a short panel stru cture we are not able to use alternative estimation methods (e.g. a fixed effects estimator or a GMM first-differen ce estimator).[START_REF] Arellano | Some Tests of Specification for Panel Data: Monte Carlo Evidence and an Application to Employment Equations[END_REF] explain that the GMM firstdifferen ce estimator requires two or more lags of all the right-hand-side variables as instruments.

Region dummies are Bangkok and Metropolitan area, Central, East, North and South (see Table 8 in the Appendix).

Due to limitations of space we do not include the methodology underlying our[START_REF] Levinsohn | Estimation Production Functions Using Inputs to Control for Unobservables[END_REF] and[START_REF] Buettner | R&D and the Dynamics of Productivity[END_REF] TFP calculations but this information is available from the authors upon request.

Horizontal FDI takes place when fo reign firms establish production plants with similar production activities in different countries to serve lo cal and neighbouring markets. Vertical FDI is where fo reign firms locate different stages of p roduction in other countries that can p roduce at a lower cost.

[START_REF] Buettner | R&D and the Dynamics of Productivity[END_REF] measure of TFP w e lose approximately four percent o f our observations.

( 1) it

We gratefully acknowledge the support of Leverhulme Trust grant number F/00094/AG and useful comments from Eric Strobl, Marius Brülhart, Matthew Cole and an anonymous referee.

The usual disclaimer applies.

Appendix

Count data for the number of products produced by each firm.

( 1) it

EX

A dummy variable for export status where a dummy equals 1 if firm i has positive export sales and 0 otherwise.

( 1) it FOREIGN A dummy variable that indicates the structure of foreign ownership where a dummy equals 1 if shares of at least 10% are foreign owned.

( 1) 25 it FOREIGN A dummy variable that indicates the structure of foreign ownership where a dummy equals 1 if shares of at least 25% are foreign owned.

( 1) 50 it FOREIGN A dummy variable that indicates the structure of foreign ownership where a dummy equals 1 if shares of at least 50% are foreign owned.

( 1)

LP it TFP

Total factor productivity that is obtained from the estimation of the semi-parametric approach of [START_REF] Levinsohn | Estimation Production Functions Using Inputs to Control for Unobservables[END_REF].

( 1) BUETTNER it TFP Total factor productivity obtained from the system estimation (a semiparametric and nonlinear least square regression) by [START_REF] Buettner | R&D and the Dynamics of Productivity[END_REF].

( 1)

LABPRO D it TFP

Labour productivity calculated as the log of value added divided by total labour.

( 1) it size Size is measured as the log of total employees.

( 1) it SMALL A dummy variable equal to 1 if the total labour of firm i at time 1 t is in the first quartile of the distribution of the total labour of all firms operating in the same 2-digit ISIC level (Rev. 3) as firm i at time 1 t .

( 1) it

LARGE

A dummy variable equal to 1 if the total labour of firm i at time 1 t is in the third quartile of the distribution of the total labour of all firms operating in the same 2-digit ISIC level (Rev. 3) as firm i at time 1 t .

( 1) it

VLARGE

A dummy variable equal to 1 if the total labour of the firm i at time 1 t is in the forth quartile of the distribution of the total labour of all firms operating in the same 2-digit ISIC level (Rev. 3) as firm i at time 1 t .

( 1) it w age

The log of wage per employee calculated as the ratio of total labour payments over total labour less owner's wage.

( 1) it RDPRODUCT A dummy variable equals 1 if a firm carries out R&D in product development and 0 otherwise.


Markus M Simon
email: simon@immunbio.mpg.de

Lars T Joeckel

Reinhard Wallich

Praxedis Julián Pardo

Praxedis Martin

Christoph Borner

Christopher Froelich

Stefan F Martin

Felix C Weber

Diego Sánchez

Diego Sanchez- Martinez

la diffusion de documents scientifiques de niveau

Keywords: T killer cell, virus control, orphan granzymes, cell death, inflammation gzm, granzyme, Hu, human, IL-1β, Interleukin-1 beta, IL-1Ra, IL-1 receptor antagonist (Anakinra), i.p., intraperitoneal, ko, knock out, LCMV, Lymphocytic Choriomeningitis virus, MEF, mouse embryonic fibroblast, Mo, mouse, NK, Natural Killer, PEMØ, peritoneal macrophage, perf, perforin, p.i., post infection, rec., recombinant, SET, nucleosome assembly protein, SLO, streptolysin O, SN, supernatant, Tc: T killer cell, wt, wild type

recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction:

The cytotoxic cell granule secretory pathway is considered indispensable for eliminating tumor and virally infected cells. The underlying process is mediated by the pore-forming protein, perforin (perf), which delivers a family of serine proteases, namely the granzymes (gzms), to cells, presumably targeted for destruction. The gzms have been characterized by DNA sequencing of counterparts in mouse, rat and man and by the availability of recombinant proteins for functional and structural analyses 1 2 . Based on their transfer into target cells and secretion extracellularly, gzms were originally anticipated to act both intracellularly and mediate extracellular proteolytic events [START_REF] Kramer | Are Proteinases Functional Molecules of T Cells?[END_REF] . However the discovery that isolated gzms induced apoptosis in the presence of perf [START_REF] Hayes | Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A[END_REF][START_REF] Shi | A natural killer cell granule protein that induces DNA fragmentation and apoptosis[END_REF] encouraged the notion that the proteases acted primarily as death effectors.

Human (Hu)gzmA was reported to induce pro-inflammatory cytokines in human monocytes and mouse macrophages more than 10 years ago [START_REF] Sower | Extracellular activities of human granzyme A. Monocyte activation by granzyme A versus alpha-thrombin[END_REF] which may be due to processing of interleukin (IL) -1 [START_REF] Irmler | Granzyme A is an interleukin 1 beta-converting enzyme[END_REF] . The significance of this observation was not appreciated until recently when human-and mouse (Mo)gzmA were observed to lack cytotoxic activity in vitro [START_REF] Metkar | Human and mouse granzyme A induce a proinflammatory cytokine response[END_REF] except under very restricted conditions [START_REF] Metkar | Human and mouse granzyme A induce a proinflammatory cytokine response[END_REF][START_REF] Beresford | Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation[END_REF] . In the absence of the apparent cytotoxicity mediated by human-and (Mo)gzmA, the possibility that the protease might act in vivo to induce pro-inflammatory cytokines was re-evaluated.

In this regard, isolated human-and (Mo)gzmA as well as human Natural killer (NK) and mouse T killer cells secreting the protease were shown to induce human monocytic cells or pre-sensitized mouse peritoneal macrophages PEMØ, respectively, to express and secrete the pro-inflammatory cytokines IL-1β, tumor necrosis factor (TNF)-α and/or IL-6 [START_REF] Metkar | Human and mouse granzyme A induce a proinflammatory cytokine response[END_REF] . In addition, caspase-1 inhibition was found to reduce (Hu)gzmA-induced IL-1β and TNF-α secretion by stimulated human monocytes suggesting that gzmA may be another activator of the inflammasome platform systems [START_REF] Mariathasan | Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation[END_REF] . The physiological importance of this phenomenon was then validated by showing that gzmA knockout (ko) mice (gzmA -/-) resist the lethal effects of LPS [START_REF] Metkar | Human and mouse granzyme A induce a proinflammatory cytokine response[END_REF] . Together with the recent finding that (Mo)gzmM augments TLR4-driven inflammation and endotoxicosis [START_REF] Anthony | A role for granzyme M in TLR4-driven inflammation and endotoxicosis[END_REF] , these observations set a biological precedent indicating that (Hu/Mo)gzms may have additional functions besides acting as proapoptotic mediators.

The highly cationic gzmK from human and mouse has tryptase-like substrate preference similar to (Hu)gzmA but the fine specificity is undoubtedly unique [START_REF] Bovenschen | Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A[END_REF] .

Similar to (Hu)gzmA, the original report indicated that isolated Rat and (Hu)gzmK are cytotoxic in vitro [START_REF] Shi | Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions[END_REF] . A more recent study suggests that isolated (Hu)gzmK induces biochemical events similar to (Hu)gzmA in target cells. Here (Hu)gzmK processes the nucleosome assembly protein (SET) releasing the DNAse NM23H1, which translocates to the nucleus. The apurinic endonuclease 1 (Ape-1) is also cleaved by (Hu)gzmK. On the other hand, caspase-3 activation is undetectable [START_REF] Zhao | Granzyme K directly processes Bid to release cytochrome c and endonuclease G leading to mitochondria-dependent cell death[END_REF][START_REF] Zhao | Granzyme K cleaves the nucleosome assembly protein SET to induce single-stranded DNA nicks of target cells[END_REF][START_REF] Guo | Granzyme K degrades the redox/DNA repair enzyme Ape1 to trigger oxidative stress of target cells leading to cytotoxicity[END_REF] . (Hu)gzmK may also cleave the tumor suppressor, p53, sensitizing tumor cells for apoptosis induction [START_REF] Hua | Ignition of p53 bomb sensitizes tumor cells to granzyme K-mediated cytolysis[END_REF] and process a vasolin-containing protein contributes to endoplasmic reticulum stress and caspase-independent cytotoxicity [START_REF] Guo | Valosin-containing protein cleavage by granzyme K accelerates an endoplasmic reticulum stress leading to caspase-independent cytotoxicity of target tumor cells[END_REF] . Compared to the proposed cytotoxic role for gzmK, the protease has been found by immunoassays [START_REF] Bade | Detection of soluble human granzyme K in vitro and in vivo[END_REF] to be elevated in the plasma of patients with sepsis [START_REF] Rucevic | Altered Levels and Molecular Forms of Granzyme K in Plasma from Septic Patients[END_REF] and in the bronchoalveolar fluid of patients with asthma and viral pneumonias [START_REF] Bratke | Granzyme K: a novel mediator in acute airway inflammation[END_REF] suggesting that gzmK may have additional biological functions.

Perf -/-mice demonstrate the critical role of the granule-secretory pathway in the recovery from LCMV infection [START_REF] Kagi | Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice[END_REF] . However, compared to perf, the contribution of gzmA, gzmB and orphan gzms in the host defense against this pathogen is controversial [START_REF] Balkow | Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection[END_REF][START_REF] Zajac | Control of lymphocytic choriomeningitis virus infection in granzyme B deficient mice[END_REF] . After intraperitoneal (i.p.) challenge with LCMV, gzmB -/-and gzmAxB -/-mice clear the virus albeit somewhat less vigorously than wild type (wt) mice. A recent study showed that (Mo)gzmK is expressed as mRNA by ex vivo LCMV-immune Tc cells, independent of their expression of gzmA and/or gzmB [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] .

GzmK has been suggested to contribute to the clearance of influenza virus in mice [START_REF] Jenkins | Heterogeneity of effector phenotype for acute phase and memory influenza A virus-specific CTL[END_REF][START_REF] Jenkins | Granzyme K Expressing Cytotoxic T Lymphocytes Protects Against Influenza Virus in Granzyme AB(/) Mice[END_REF] but overall the biological function(s) of this gzm family member remains incompletely characterized. The purpose of this report is to re-examine the cytotoxic activity of (Mo)gzmK and, due to its similar substrate specificity to (Hu/Mo)gzmA, determine whether the protease has pro-inflammatory effects.

Results:

LCMV infection in mice is readily controlled in the absence of gzmA and B Previous studies demonstrated that although perf is essential for optimal control of LCMV infection [START_REF] Kagi | Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice[END_REF] , gzmA and B play a marginal role [START_REF] Balkow | Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection[END_REF][START_REF] Zajac | Control of lymphocytic choriomeningitis virus infection in granzyme B deficient mice[END_REF] . To verify this supposition, we compared survival and viral titers in mice lacking gzmA and B (gzmAxB -/-) to those without perf and the two gzms (perfxgzmAxB -/-). After challenge with 1x10 [START_REF] Shi | A natural killer cell granule protein that induces DNA fragmentation and apoptosis[END_REF] PFU LCMV-WE, all perfxgzmAxB -/-(9/9) died but only 1/9 gzmAxB -/-and none of the wild type (wt) B6 mice succumbed to the virus during the 30 day observation period (Figure 1A). At day eight after inoculation, hepatic virus titers were similar increased in the WT as well as the gzmAxB -/-and triple ko mice with somewhat higher levels in the ko mouse strains (Figure 1B). However, while the level of virus gradually declined to background levels in gzmAxB -/-and B6 mice, no reduction of virus load was observed in the liver of perfxgzmAxB -/-mice during the entire observation period (Figure 1B). The data are consistent with previous studies [START_REF] Balkow | Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection[END_REF] emphasizing that the control of LCMV infection, including viral elimination, is strictly dependent on perf but that neither gzmA nor gzmB are obligatory participants.

Granzyme K is expressed in LCMV-immune Tc cells from wild-type and gzmAxB-deficient mice

To test the assumption that other gzms beside gzmA and B might contribute to perfmediated control of LCMV infection in gzmAxB -/-mice, virus-immune Tc cells (d 8 p.i.) were evaluated for the expression of perf and gzm-specific mRNAs and their respective intracellular proteins. As reported previously [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] , virus-immune Tc cell from B6, gzmAxB -/-and perfxgzmAxB -/-mice expressed similar levels of the gzmK transcript as well as the mRNA for gzmA, gzmB and perf expected for the respective ko mice (Figure 1C). No transcripts for gzmC-G and gzmM were detectable (for gzmM see also Supp. Fig. 1C). To ensure that the various effector populations expressed gzmK protein, its presence in CD8 + Tc cells of uninfected and LCMV infected mice (day 8 p.i.) was determined using a recently developed rabbit antirec(Mo)gzmK antibody. Although gzmK was undetectable in Tc cells from noninfected mice, Tc cells from all three infected mouse strains contained similar levels of the protease (Figure 1D). As expected, only Tc cells from LCMV immune B6 but not gzmAxB -/-and perfxgzmAxB -/-mice expressed the gzmA and gzmB proteins (data not shown).

LCMV-immune Tc cells that express gzmK and lack gzmA and gzmB are noncytotoxic

To determine whether LCMV-immune gzmAxB -/-Tc cells that expressed primarily gzmK induce apoptosis in MEF cells, we measured multiple parameters including PS externalization, PI uptake, mitochondrial depolarization (ΔΨ m ) and ROS generation.

Despite the fact that LCMV-immune B6 and gzmAxB -/-mice both readily controlled LCMV infection, only ex vivo derived LCMV-immune B6 Tc cells induced cell death in target cells in vitro as defined by the abovementioned parameters (Figure 2A).

LCMV-immune gzmAxB -/-Tc cells, on the other hand, were only marginally cytotoxic while LCMV-immune perfxgzmAxB -/-Tc cells were completely inactive. On the other hand, LCMV-immune gzmAxB -/-NK cells, which expressed perf and gzmM, but no gzmK, showed residual cytotoxic activiy which was however at least five times lower than those of gzmA -/-mice (Supp. Figure 1A).

Recombinant mouse gzmK and gzmA are not cytotoxic

To compare the cytotoxic potential of isolated rec(Mo)gzmK with rec(Mo)gzmA and B, EL4 cells were incubated with increasing concentrations of the proteases (150-1200nM) delivered by SLO and cell death was determined by PI uptake, annexin reactivity (PS exposure), Δψ, and ROS generation. Although neither (Mo)gzmA nor (Mo)gzmK elicited a cytotoxic response at all concentration, (Mo)gzmB showed the predicted concentration-dependent induction of cell death (Figure 2B), including cleavage of caspase 3 (Figure 2C). To ensure that rec(Mo)gzmA and rec(Mo)gzmK manifested enzymatic activity and to verify their predicted differences in cleavage specificity [START_REF] Bovenschen | Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A[END_REF] , their amidolytic activities were compared against four different chromogenic substrates. Both gzms were proteolytically active with each exhibiting distinct preferences for the four related chromogenic substrates, especially Tos-Glypro-Arg-pNA (Table I). Finally, a cell survival assay was performed [START_REF] Pardo | Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin[END_REF] to exclude the possibility that gzmK might induce cell death that is undetected by the parameters mentioned above. Re-growth of EL4 cells was unimpaired for targets incubated only with the rec(Mo)gzmK (1000 nM) or after the protease was delivered by SLO (Figure 2D).

Recombinant mouse granzyme K induces macrophages to release IL-1β

Although rec(Mo)gzmA and rec(Mo)gzmK have different substrate specificities, we speculated that the tryptase-like activity of (Mo)gzmK might mimic (Mo)gzmA in its capacity to induce pre-activated PEMØ to express and secrete mature IL-1β. In this regard, rec(Mo)gzmK was added to LPS-primed PEMØ in the presence or absence SLO. The delivery of gzmK by SLO induces concentration-dependent processing of pro-IL-1β, requiring doses above 30 nM (Figure 3A and Suppl. Figure 2). SLO alone was observed to also induce detectable albeit minor maturation/secretion of IL-1β (Figure 3A and Suppl. Figure 2). At higher concentrations of gzmK (600 and 1000 nM), the protease alone was sufficient to induce release of IL-1β to levels observed for cells treated with the granzyme and SLO. Finally, the inactive pro-form of rec(Mo)gzmK did not alter maturation/secretion of IL-1β (Supp. Figure 2) eliminating the possibility that the granzyme was inducing the cytokine through a non-proteolytic mechanism.

LCMV-immune Tc cells of B6 and gzmAxB -/-mice induce IL-1β release from wt and P2X 7

-/-macrophages LCMV-immune B6 and gzmAxB -/-Tc cells were added to LPS-primed PEMØ in the presence or absence of LCMV-specific immunogenic peptide, gp33. In the absence of gp33, LCMV-immune B6 and gzmAxB -/-Tc cells induced only marginal processing and release of IL-1β in PEMØ at an E:T ratio of 5:1. In contrast, gp33 pulsed targets responded to LCMV-immune B6 as well as gzmAxB -/-Tc cells by secreting substantial amounts of IL-1β (p17; Figure 3B). To exclude the possibility that the Tc cell-induced IL-1β secretion was due to ATP released from stressed PEMØ [START_REF] Surprenant | Signaling at purinergic P2X receptors[END_REF][START_REF] Martinon | The inflammasomes: guardians of the body[END_REF] , the response of cells from mice lacking the ATP sensor (P2X7 receptor) were used under similar conditions. While treatment with ATP (5mM) did not result in secretion of active IL-1β, wt and gzmAxB -/-Tc cells readily induced the release of mature IL-1β from P2X7-deficient PEMØ to an extend similar to wt PEMØ. (Figure 3C).

In vivo treatment of mice with IL-1RA inhibits virus elimination

To explore the possibility that IL-β participates in the control of LCMV infection, B6 mice were treated with IL-1Ra (Anakinra; [START_REF] So | A pilot study of IL-1 inhibition by anakinra in acute gout[END_REF] ) and virus titers were monitored in liver on days 8, 12 and 19. In contrast to mock-treated and virus-infected B6 mice (Figure 1B), recipients treated with Anakinra were unable to eliminate LCMV (Figure 4A).

This outcome was not due to the absence of activated Tc because the ratio of LCMV specific Tc is comparable for untreated and treated LCMV infected mice (Figure 4B).

Furthermore, LCMV-immune Tc taken from the same Anakinra treated mice showed pro-apoptotic activity similar to mock-treated Tc (Figure 4C).

Discussion:

The observation that perf -/-mice die from LCMV infection clearly indicates that the pore forming protein perf is essential for Tc cell (NK)-mediated control of this virus [START_REF] Kagi | Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice[END_REF] .

However, since perf functions chiefly to deliver gzms and other granule components into target cells, an understanding of the role(s) played by these proteins in the antiviral response and associated tissue damage is crucial. For example, LCMV is cleared in gzmA -/-and gzmB -/-mice although recovery is delayed in the latter compared to wt controls [START_REF] Zajac | Control of lymphocytic choriomeningitis virus infection in granzyme B deficient mice[END_REF] and LCMV-associated hepatitis appears to require the presence of gzmA and gzmB [START_REF] Balkow | Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection[END_REF] . We have observed here that the course of virus infection was similar, but not identical for gzmAxB -/-and B6 mice indicating that other granule proteins are possible participants [START_REF] Grossman | The orphan granzymes of humans and mice[END_REF] . Since Tc cells are essential to control LCMV infection and gzmK is expressed in virus-immune gzmAxB -/-Tc cells that retain this capacity, one could speculate that gzmK together with perf may contribute to the host response that results in the elimination of the virus.

Although the molecular basis for a gzmK-mediated anti-viral response is uncertain, based on past observations, the tryptase is predicted to clear LCMV by inducing infected cells to undergo apoptosis. By studying the human system, the Fan group has shown that rec(Hu)gzmK elicits multiple pro-apoptotic pathways that could participate in anti-viral immunity. Their lab has reported that the granzyme directly processes Bid which releases cytochrome c and endonuclease G, resulting in the activation of a mitochondrial-dependent cell death pathway albeit without caspase 3 activation [START_REF] Zhao | Granzyme K directly processes Bid to release cytochrome c and endonuclease G leading to mitochondria-dependent cell death[END_REF] . Rec(Hu)gzmK, similar to rec(Hu)gzmA, has also been reported to cleave SET which occurs in association with single-stranded DNA nicks in target cells [START_REF] Zhao | Granzyme K cleaves the nucleosome assembly protein SET to induce single-stranded DNA nicks of target cells[END_REF] . Furthermore, rec(Hu)gzmK has been reported to cleave p53 into three products (p40, p35, and p13) which apparently possess pro-apoptotic activities. In addition, these fragments are proposed to augment the cytotoxic activity of gzmK, though other biological effects cannot be excluded [START_REF] Hua | Ignition of p53 bomb sensitizes tumor cells to granzyme K-mediated cytolysis[END_REF] . Finally, (Hu)gzmK was shown to cleave a vasolin-containing protein resulting in accelerated endoplasmic reticulum stress and caspase-independent cytotoxicity of tumor cells [START_REF] Guo | Valosin-containing protein cleavage by granzyme K accelerates an endoplasmic reticulum stress leading to caspase-independent cytotoxicity of target tumor cells[END_REF] . In marked contrast, the present study demonstrates that ex vivo-derived gzmK + but gzmAxB -/-LCMV-immune Tc cells expressed only marginal pro-apoptotic/ cytotoxic activities especially when measured by the sensitive markers that assess plasma membrane and mitochondrial integrity as well as by a target cell survival assay. This finding agrees with reports

showing that LCMV-and ectromelia-immune gzmAxB -/-Tc cells have marginal proapoptotic activity [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF][START_REF] Pardo | Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTLmediated target cell lysis[END_REF][START_REF] Pardo | The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation[END_REF] . More strikingly, rec(Mo)gzmK, like (Mo)gzmA [START_REF] Metkar | Human and mouse granzyme A induce a proinflammatory cytokine response[END_REF] , completely failed to induce cell death at concentrations that suffice for gzmB-induced apoptosis.

The combined results suggest that MogzmK, through non-cytotoxic processes, might be one component in the complex of molecular events that contribute to elimination of LCMV. Notably, NK cells from infected gzmAxB -/-mice, which did not express gzmK as expected from previous studies [START_REF] Chowdhury | Death by a thousand cuts: granzyme pathways of programmed cell death[END_REF] showed low but significant cytotoxic activity, which may be due to the expression of gzmM. However, the possibility that LCMV infection in these ko mice is controlled by NK cells is less likely, since NK cells have been shown before to be unable on their own to control LCMV infection, even if they fully express the exocytosis pathway.

Offering a major paradigm shift in characterizing the functionality of the granule secretory pathway, we have learned that isolated (Mo)gzmA and ex vivo virusimmune mouse Tc cells containing gzmA induce IL-1β release from LPS-sensitized PEMØ [START_REF] Metkar | Human and mouse granzyme A induce a proinflammatory cytokine response[END_REF][START_REF] Froelich | Granule-associated serine proteases: granzymes might not just be killer proteases[END_REF] . Since gzmA may contribute to host defense by inducing pro-inflammatory cytokines in pathogen-infected cells targeted by the granule secretory process, we speculated that (Mo)gzmK, displaying a tryptase-like activity similar but not identical to (Mo)gzmA [START_REF] Bovenschen | Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A[END_REF] , might also be an inducer of pro-inflammatory cytokines. Accordingly, we show here for the first time that nanomolar concentrations of rec(Mo)gzmK and LCMV-immune Tc containing gzmK but neither gzmA nor gzmB induce the maturation and secretion of IL-1β in LPS-sensitized PEMØ. The fact that gzmK displays a highly restricted substrate specificity that only partially overlaps with (Mo)gzmA [START_REF] Bovenschen | Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A[END_REF] , suggests that (Mo)gzmK may augment (Mo)gzmA-induced proinflammatory processes by differentially cleaving the same or distinct specific substrates. This assumption is supported by the fact that B6 Tc cells, which express gzmA and gzmK together with gzmB, have a greater capacity than gzmAxB -/-Tc cells to induce IL-1β release from LPS-sensitized PEMØ.

Pro-IL-1β is produced in response to danger or pathogen associated molecular patterns like LPS and can be processed to the mature and secreted cytokine by the NLRP3 inflammasome, a caspase-1 activating cytosolic protein complex [START_REF] Martinon | The inflammasomes: guardians of the body[END_REF] . The NLRP3 inflammasome is activated by numerous stimuli such as bacterial toxins, uric acid crystals but also by extracellular ATP released from stressed or damaged cells.

This endogenous danger signal is sensed by the purinergic receptor P2X 7 , a ligand gated ion channel [START_REF] Surprenant | Signaling at purinergic P2X receptors[END_REF] . Our results show that the release of IL-1β of PEMØ induced by B6 as well as gzmAxB -/-Tc was not affected by the absence of P2X 7 receptor. This result suggests that the Tc-induced IL-1β processing is not due to ATP derived from damaged target cells and therefore strengthening the concept that pro-inflammatory granzymes such as gzmK participate in inflammasome activation.

Overall, the data show that neither gzmA nor gzmB are essential to control LCMV infection in mice. This observation, which supports previous work [START_REF] Balkow | Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection[END_REF][START_REF] Zajac | Control of lymphocytic choriomeningitis virus infection in granzyme B deficient mice[END_REF] , is intriguing especially when combined with results here indicating ex vivo anti-LCMV Tc cells from gzmAxB -/-mice exhibit markedly reduced cytotoxicity in vitro. Similar to these Tc which contain primarily (Mo)gzmK, rec(Mo)gzmK delivered by SLO also failed to mount the anticipated cytotoxic response. Thus, the anti-viral effects of in vivo anti-LCMV Tc cells does not correlate with their in vitro cytolytic activity, but rather is mediated through non-cytotoxic pathways such as inflammasome activation or uncharacterized biological effects [START_REF] Andrade | Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition[END_REF] .

The possibility that (Mo)gzmK induces IL-1β release which aids LCMV clearance is further supported by the observation that mice treated with Anakinra are unable to control replication, even though the ratio of LCMV-immune Tc cells generated under these conditions, including their cytotoxic potential was similar to those of untreated infected B6 mice (Figure 4B,C). These findings differ from a recent study where IL-1R -/-mice, challenged with influenza virus, were unable to generate virus-specific Tc

37

. This discrepancy may be due to the different approaches used to reduce IL-1β in the two systems. In the IL-1R -/-mice, IL-1β is expected to be unable to mediate its myriad biological effects through the IL-1R including the induction of Tc. The blockade produced by Anakinra is predicted to be incomplete allowing the induction of virus-specific Tc cells but curtailing the anti-viral activity capacity of the cytokine during the effector phase of the host response. Although (Mo)gzmK, similar to (Hu)gzmH [START_REF] Andrade | Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition[END_REF] , might mediate direct antiviral effects that contribute to viral clearance, together the evidence supports the existence of a (Mo)gzmK-induced proinflammatory pathway that controls intracellular pathogens.

In comparison to (Mo)gzmK, isolated (Hu)gzmK has been reported to be directly cytotoxic in vitro when delivered by SLO or adenoviral particles [START_REF] Zhao | Granzyme K directly processes Bid to release cytochrome c and endonuclease G leading to mitochondria-dependent cell death[END_REF] . However, others have been unable to demonstrate the rec(Hu)gzmk is cytotoxic in vitro when delivered by SLO to Hela cell targets at concentrations reaching 1000 nM (personal communication, Niels Bovenschen). Using intact Tc containing primarily (Mo)gzmK, we have verified that (Mo)gzmK delivered in vitro is not cytotoxic. A similar approach will be necessary to reconcile whether (Hu)gzmK is cytotoxic.

Future studies that utilize gzmK-deficient mice will be necessary to verify whether LCMV infection is controlled by gzmK and/or other granule-associated components and whether the putative anti-viral activity of gzmK is linked to the induction of proinflammatory cytokines in macrophage subsets or to other non-cytotoxic pathways. In contrast to LCMV, other viruses such as ectromelia depend on cytolytic gzmB [START_REF] Mullbacher | Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes[END_REF] .

Thus, depending of the virus and the tissue distribution of infection, Tc cells may use individual gzms to kill infected targets and employ one or more of the gzms to disrupt their life cycle by non-cytolytic means.

Materials and Methods:

Mouse strains -The following strains were maintained at the Max-Planck-Institut für Immunobiologie, Freiburg: C57BL/6 (B6; wild type, wt); strains deficient for gzmA (gzmA -/-), gzmA and gzmB (gzmAxB -/-) and perf, gzmA and gzmB deficient (perfxgzmAxB -/-), were bred on the B6 background [START_REF] Balkow | Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection[END_REF] . Mice deficent for P2X 7 (P2X was expanded in L929 fibroblasts, as described [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] . The virus-specific epitope gp33

(KAVYNFATATC; Neosystem, Strasbourg, France) for H-2D b was used for target labeling in cytotoxicity assays [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] .

Disease model -Mice were infected intraperitoneally (i.p.) with 10 5 PFU of LCMV-WE according to established protocols [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF][START_REF] Rode | Perforin and Fas act together in the induction of apoptosis, and both are critical in the clearance of lymphocytic choriomeningitis virus infection[END_REF] .

IL-1Ra (Anakinra) treatment -Mice were injected 1 day before infection with 10 5

PFU LCMV-WE with 200 µg IL-1Ra (Anakinra, Kineret ® Amgen, Breda, Netherlands) in 200 µl PBS and from then on every third day with the same dose until the end of experiment [START_REF] So | A pilot study of IL-1 inhibition by anakinra in acute gout[END_REF] .

Virus titers -Aliquots of liver tissues were homogenized and used for determination of virus titers as described elsewhere [START_REF] Rode | Perforin and Fas act together in the induction of apoptosis, and both are critical in the clearance of lymphocytic choriomeningitis virus infection[END_REF] .

Generation of ex vivo CD8

+ cells -On day eight post infection (p.i.), CD8 + cells were positively selected from spleen using α-CD8-MicroBeads (Miltenyi Biotec Bergisch Gladbach, Germany) with an autoMACS (Milteny Biotec, Bergisch Gladbach, Germany) and resuspended in MEM/5%FCS prior to use in cytotoxic assays as described [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] . Detection of LCMV-immune CTL was conducted by double staining with CD8 antibody (BD Pharmingen, San Diego, CA, USA ; clone 53-67) and gp33-labeled pentamers (ProImmune, Oxford, UK) as described [START_REF] Pardo | Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTLmediated target cell lysis[END_REF] .

Cell lines, cell culture and reagents -The mouse cell lines 1.3E6 (Tc cell line) and EL4.F15 (thymoma) and mouse embryonic fibroblast (MEF) cell lines were cultured as described [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF][START_REF] Martin | Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes?[END_REF] . Recombinant (rec.) (Mo)gzmK was produced in E.coli B834 (DE3) using the pET-21a vector and purified as described [START_REF] Wilharm | Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma[END_REF] . Rabbit immune serum (IS) specific for (Mo)gzmK was generated as described for (Mo)gzmB [START_REF] Pardo | Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin[END_REF] .

Analysis of pro-apoptotic processes -Cell death induced by ex vivo CD8-enriched

Tc cells or by isolated gzms in the presence or absence of Streptolysin O (SLO), was analyzed as described [START_REF] Pardo | Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTLmediated target cell lysis[END_REF] . Briefly, target cells were pre-treated with LCMVimmunodominant peptide gp33 for 2 h prior to incubation with ex vivo-derived LCMVimmune CTL at 10:1 E:T cell ratio for 4h at 37°C, 7% CO 2 . Alternatively, target cells were incubated with the indicated concentration of rec.gzms +/-SLO for 4h.

Subsequently, different apoptotic parameters were tested in the target population (CD8 -) by FACS with a FACSCalibur (BD Pharmingen, San Diego, CA, USA) and FLOWJO ® (Ashland, OR, USA) software as described [START_REF] Metkar | Human and mouse granzyme A induce a proinflammatory cytokine response[END_REF][START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] . In short:

Cell membrane and mitochondrial membrane perturbations: Phosphatidylserine (PS) Intracellular staining -Cell populations were analyzed for cell surface markers and intracellular gzm expression as described elsewhere [START_REF] Martin | Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes?[END_REF] , analyzed by FACS with a FACSCalibur (BD Pharmingen San Diego, CA, USA) and FLOWJO ® (Ashland, OR, USA) software.

Survival assay -Survival of target cells following their incubation with rec(Mo)gzmK in presence and absence of SLO was analyzed as described [START_REF] Pardo | The differential contribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated apoptosis is determined by the quality of target cells[END_REF] . Briefly, target cells were incubated with 1000 nM rec(Mo)gzmK in the presence or absence of SLO for 4h. Afterwards 3 H-thymidine was added and cells were incubated for 10h at 37°C, 5%CO 2 , without removing rec(Mo)gzmK and SLO. Subsequently cells were harvested and target cell survival was quantified by 3 H-thymidine incorporation as described [START_REF] Pardo | The differential contribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated apoptosis is determined by the quality of target cells[END_REF] . This assay gives similar result to clonogenic survival assays in agar plates [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] .

Enzymatic Assays -Amidolytic activity of gzms using the following chromogenic substrates (Bachem, Weil am Rhein, Germany) was performed as described previously [START_REF] Simon | Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes[END_REF] : Bz-Arg-pNA; H-D-Pro-Phe-Arg-NA; Ac-Ile-Glu-Pro-Asp-pNA; Tos-Gly-Pro-Arg-pNA.

RT-PCR -Total RNA was extracted from up to 5 x 10 6 ex vivo CD8 + cells, using the QIAshredder spin columns, the RNAeasy Mini Kit and the RNAse-free DNAse Kit (all from Qiagen, Hilden, Germany) according to the manufacturer's instructions, and specific transcripts were amplified. The sense/ antisense primers for gzmA, gzmC, gzmD, gzmE, gzmF, gzmG and Hprt1 are described in [START_REF] Martin | Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes?[END_REF] and [START_REF] Revell | Granzyme B and the downstream granzymes C and/or F are important for cytotoxic lymphocyte functions[END_REF] , respectively.

Sense/antisense primers for gzmB, gzmK and gzmM are described in [START_REF] Pardo | Granzyme Binduced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs[END_REF] . Primers for perf are described in [START_REF] Balkow | Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection[END_REF] .

IL-1β release assay -B6 mice were injected i.p. with 2 ml of 4% thioglycollate (Sigma, Steinheim, Germany) in LPS-free water, three days prior to removal of peritoneal macrophages (PEMØ). On d3 post injection, mice were sacrificed and their peritoneum was washed with 10ml of PBS to collect PEMØ. The cellsuspension was washed and 8 x 10

Titles and legends to figures:

Figure 1. A Survival of wild-type, gzmAxB -/-and perfxgzmAxB -/-mice infected with LCMV-WE. Groups (9 mice each) of B6 (dashed line) or gzmAxB -/-(dotted line) or perfgzmAxB -/-(line) mice were infected with 1x10 5 PFU LCMV-WE i.p. and survival was monitored for 30 days.

B LCMV-WE replication in liver of WT, gzmAxB -/-and perfxgzmAxB -/-mice. Groups of B6, gzmAxB -/-and perfxgzmAxB -/-(18 mice per strain) recipients were infected with 1x10 C Anakinra treated and control Tc mediate similar apoptotic effects against EL4 cells.

Gp-33-pulsed or untreated EL4 target cells were incubated with ex vivo LCMVimmune CD8-enriched Tc cells (4h, 10:1). Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake were analysed via flow-cytometry.

Tables:

Table 1: Amidolytic activity of gzmA and gzmK on chromogenic substrates Bz-Arg-pNA 0 3

  exposure and Propidium Iodide PI uptake were analysed by FACS using the annexin-V-FITC kit from BD Pharmingen (San Diego, CA USA). The mitochondrial membrane potential was measured with the fluorescent probe 3,3'dihexyloxacarbocyanine iodide (DiOC 6 (3); (Molecular Probes Eugene, OR, USA) and Reactive Oxygen Species (ROS) generation with 2-hydroxyethidium (2-HE; Molecular Probes Eugene, OR, USA)

Figure 2 A

 2 Figure 2 A Induction of pro-apoptotic markers by ex vivo LCMV-immune Tc cells from WT, gzmAxB -/-and perfxgzmAxB -/-mice. Gp-33-pulsed or untreated MEF target cells were incubated with ex vivo LCMV-immune CD8-enriched Tc cells of the indicated strain (4h, 10:1 effector/target ratio). Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake and in parallel ΔΨ m loss (DiOC 6 ) and ROS generation (2-HE) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression as described in Materials Methods. Data in the panels are represented as the mean±S.E.M. of three independent experiments. B rec(Mo)gzmA and rec(Mo)gzmK are not cytotoxic: analyses of apoptotic parameters during gzmA, gzmB and gzmK-induced cell death in EL4 cells. EL4 cells were incubated with the indicated amounts of gzms in the presence of a sublytic

Figure 3 ABC

 3 Figure 3 A Induction of IL-1β release by rec(Mo)gzmK. Adherent B6 PEMØ, previously sensitized with thioglycollate in vivo and challenged with LPS in vitro were incubated with the indicated amounts of gzmK +/-0,25µg/ml SLO, or ATP as positive control, for 3h and SN were subjected to WB analysis using anti-IL-1β specific Ab. Aliquots of the following preparations were used as standards for WB analysis: SN of ATP sensitized pre-activated B6 PEMØ; SN of thioglycollate pre-sensitized and LPS challenged B6 PEMØ. B Induction of IL-1β release by ex vivo CD8 cells expressing gzmK. Thioglycollate pre-sensitized and LPS challenged adherent B6 PEMØ were incubated with ex vivoderived LCMV-immune Tc cells from B6 or gzmAxB ko mice at an E:T ratio of 5:1 in presence or absence of the LCMV-specific peptide gp33. SN was taken after 6h and subjected to WB analysis using anti-IL-1β specific Ab. As control, SN from thioglycollate pre-sensitized and LPS challenged B6 PEMØ, and subsequently stimulated with 5mM ATP, was analysed accordingly. Aliquots of the following preparations were used as standards for WB analysis: rec.IL-1β, p17; SN of ATP sensitized pre-activated wt B6 PEMØ; SN of wt thioglycollate pre-sensitized and LPS activated B6 PEMØ. C Induction of IL-1β release by ex vivo CD8 cells expressing gzmK by targerts lacking the P2X7 receptor. Experimental procedure as in figure 3B but with macrophages from P2X 7 -/-mice. Controls were prepared individually for this experiment with P2X 7 -/-macrophages (SN of ATP sensitized pre-activated PEMØ; SN of wt thioglycollate pre-sensitized and LPS activated PEMØ).

Figure 4 AB

 4 Figure 4 A Anakinra inhibits recovery from LCMV infection. B6 mice received Anakinra before infection (1x10 5 PFU LCMV-WE i.p) and every third day thereafter. Three mice from each group were sacrificed at 8, 12 and 19 days and hepatic viral replication determined. B Anakinra treated mice infected with LCMV express levels of LCMV specific Tc cells similar to untreated controls. Mice were first treated eith Anakinra (200 µg) one day before infection and then every third day. After sacrifice (day 8), splenic CD8 cells were isolaed and virus specific Tc were determined by pentamer staining. Livers from these mice were used to generate data reported in figure 4A.

  [START_REF] Shi | A natural killer cell granule protein that induces DNA fragmentation and apoptosis[END_REF] PFU LCMV-WE i.p.. 6 mice from each group were sacrificed at 8, 12 and 19 days p. i.. Virus titers of individual mice were determined by plaque titer assay as described in Materials and Methods.

C Expression of mRNA in ex vivo LCMV-immune CD8-enriched Tc cells from WT, gzmAxB -/-and perfxgzmAxB -/-mice. Total mRNA was isolated from ex vivo LCMVimmune CD8-enriched Tc cells (d8 p.i.) from wild-type, gzmAxB -/-and perfxgzmAxB -/- mice, previously infected with 1x10 5 PFU LCMV-WE i.p. Samples were analyzed by RT-PCR for their expression of transcripts using specific primers for gzmA-G, gzmK, gzmM, Prf1 and Hprt1. D Intracellular expression of gzm and perf protein in ex vivo LCMV-immune CD8enriched Tc cells (d8 p.i.). Ex vivo LCMV-immune CD8-enriched Tc cells (d8 p.i.) of wt, gzmAxB -/-and perfxgzmAxB -/-mice, infected with 1x10 5 PFU LCMV-WE i.p. were analyzed by FACS for intracellular expression of gzmK (blue line) using the respective rabbit anti-K immune serum and the control pre-immune serum (red line).

Conflict of interest:

The authors declare no conflict of interest

Supplementary information:

Supplementary information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd) Footnotes:

1.) Address correspondence and reprint requests to: MM