Natalie R Hall
email: natalie.hall@reading.ac.uk

Richard J Crisp
email: r.crisp@kent.ac.uk.

Assimilation and contrast to group primes: The moderating role of ingroup identification

Keywords: INGROUP IDENTIFICATION, CONTRAST, SOCIAL COMPARISON, AUTOMATIC BEHAVIOR

This research examined the conditions under which behavioral contrast would be observed in relation to ingroup and outgroup primes. The authors tested the hypothesis that differing levels of commitment to the ingroup would predict diverging behavioral responses to outgroup but not ingroup primes. Across two studies, featuring both age and gender groups, we found that ingroup identification predicted responses to outgroup primes with higher identifiers showing an increased tendency to contrast, that is, behave less like the outgroup, and more like the ingroup. Ingroup identification did not predict responses to ingroup primes. The implications of these findings for social comparison and social identity theories are discussed.

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Assimilation and contrast to group primes:

The moderating role of ingroup identification Some groups matter to some people more than others. When they do, there is considerable evidence that people self-stereotype. That is, we come to see ourselves as a group member more than an individual; we more readily accept, adopt and express the attitudes and opinions that we believe our group to hold. In this article we explore the most minimal conditions needed to invoke self-stereotyping at a behavioral level. We argue that it is outgroups, not ingroups, that drive tendencies to depersonalize because they invoke social comparison tendencies; but furthermore that this will only apply for people who highly identify with their ingroup, those people for whom social comparison will be critically important for self-definition.

Social comparison and social identity

Early writings on social comparison suggested that people have a fundamental need to compare themselves with others (Festinger, 1954) and this desire to learn about the self through comparison with others is considered universal [START_REF] Gibbons | Individual differences in social comparison: Development of a scale of Social Comparison Orientation[END_REF]. The primary goal of such social comparison is to acquire information about the self (Festinger, 1954;[START_REF] Gibbons | Individual differences in social comparison: Development of a scale of Social Comparison Orientation[END_REF]Stapel & Tesser, 2001). Not all social comparisons, however, will provide the perceiver with the same information about themselves. The perceiver's impressions of their own abilities are altered by exposure to different people, for example, asked to compare their athletic abilities with Michael Jordan people report themselves to be less athletic than those asked to compare themselves with Bill Clinton [START_REF] Mussweiler | The ups and downs of social comparison: mechanisms of assimilation and contrast[END_REF].

Social comparison does not only occur on an individual level but also on a group level "Categories do not exist in isolation. A category is only such in contrast with another" (Hogg & Abrams, 1988, p.14). In the same way that perceivers use social comparison with individuals to define themselves; "[categories] provide a system of self-reference: they create and define the individuals' place in society." (Tajfel & Turner, 1979, p.40). The idea that social comparison may occur at a group level is one of the core assumptions of the social identity perspective (Tajfel &

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Outgroup Contrast 4 [START_REF] Tajfel | An integrative theory of intergroup conflict[END_REF]; including self-categorization theory, [START_REF] Turner | Rediscovering the social group: A self-categorisation theory[END_REF]. A key tenet of the social identity perspective is that self-knowledge is derived through social comparisons [START_REF] Hogg | Social identifications: A social psychology of intergroup relations and group processes[END_REF]. This means that all of an individual's perceptions both of themselves and of their group are relational and anchored in the current social context.

A salient social comparison at the group level will lead to self-categorization as an ingroup member; this will be evident in attitudinal assimilation to the group norm (Turner & Reynolds, 2001;Smith & Henry, 1996). Previous research has demonstrated that when the intergroup comparative context is salient perceivers self-categorize by self-stereotyping [START_REF] Turner | Rediscovering the social group: A self-categorisation theory[END_REF]Simon, Glassenbayer, & Stratenwerth, 1991;[START_REF] Simon | Self-Stereotyping and Social-Context -the Effects of Relative In-Group Size and In-Group Status[END_REF]Simon, Pantaleo, & Mummendey, 1995;Spears, Doosje, & Ellemers, 1997) assigning the same traits to the self and the ingroup [START_REF] Cadinu | Self-anchoring and differentiation processes in the minimal group setting[END_REF]Study 1;[START_REF] Clement | The primacy of self-referent information in perceptions of social consensus[END_REF], 2002;[START_REF] Gramzow | Memory for in-group and out-group information in a minimal group context: The self as an informational base[END_REF]Krueger & Zeiger, 1993;Mullen, Dovidio, Johnson, & Copper, 1992;[START_REF] Otten | Me" and "us" or "us" and "them"? --The self as heuristic for defining novel ingroups[END_REF], Smith, & Henry, 1996); and will tend to perceive both the ingroup and the outgroup to be more homogenous [START_REF] Ellemers | Stereotyping in social context[END_REF][START_REF] Haslam | Social categorization and group homogeneity -Changes in the perceived applicability of stereotype content as a function of comparative context and trait favorableness[END_REF]. Self-stereotyping is an expression of differentiation from outgroups, therefore it seems reasonable to draw a parallel here with research on behavioral contrast to outgroups.

Behavioral contrast to outgroup primes

Social comparison is ubiquitous (Mussweiler, 2003a;[START_REF] Mussweiler | Everything is relative': Comparison processes in social judgments. The 2002 Jaspers Lecture[END_REF] it even occurs following subliminal exposure to potential comparison standards [START_REF] Mussweiler | The ups and downs of social comparison: mechanisms of assimilation and contrast[END_REF][START_REF] Stapel | From seeing to being: Subliminal social comparisons affect implicit and explicit self-evaluations[END_REF]) and such supraliminal comparison targets have been shown to influence not only attitudes as described above, but also perceivers' subsequent behavior. The selective accessibility model (Mussweiler, 2003a;[START_REF] Mussweiler | Everything is relative': Comparison processes in social judgments. The 2002 Jaspers Lecture[END_REF] suggests that assimilative responses are observed when stimuli are perceived to be similar to a target and that contrastive responses are observed when stimuli are perceived to be different from a target. This implies that ingroup targets may be expected to lead to assimilation as they are likely to be perceived as more similar to the perceiver and outgroup

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targets are more likely to lead to contrastive responses as they are perceived to be more dissimilar to the perceiver. This prediction has received some empirical support: Ingroups have been shown to lead to assimilation of self-evaluations and behavior (Gordijn & Stapel, 2006;[START_REF] Mussweiler | I know you are, but what am I? Self evaluative consequences of judging the in-group and out-group members[END_REF] and, outgroups have been shown to lead to contrast of selfevaluations and behaviour (Gordijn & Stapel, 2006;[START_REF] Mussweiler | I know you are, but what am I? Self evaluative consequences of judging the in-group and out-group members[END_REF][START_REF] Schubert | Contrast from social stereotypes in automatic behaviour[END_REF][START_REF] Spears | Reaction in action: Intergroup contrast in automatic behavior[END_REF]. However, other research that has investigated perceivers' behavioral responses to stereotypic primes does not support the idea that ingroup primes lead to behavioral assimilation and outgroup primes lead to behavioral contrast.

Young university students have been shown to assimilate their behavior to the stereotype of the elderly by walking more slowly [START_REF] Bargh | Automaticity of social behavior: Direct effects of trait construct and stereotype activation on action[END_REF] and to the stereotype of "Professors" by performing better on a general knowledge task [START_REF] Dijksterhuis | Seeing one thing and doing another: Contrast effects in automatic behaviour[END_REF]. These findings are surprising; as other scholars have noted the perceivers in these experiments had essentially been primed with an outgroup categorization, so contrastive behavioral responses would have been predicted [START_REF] Schubert | Contrast from social stereotypes in automatic behaviour[END_REF]Spears, Gordjin, Dijksterhuis, & Stapel, 2004).

Why in Bargh and Dijksterhuis' studies did outgroup primes not lead to contrast? [START_REF] Schubert | Contrast from social stereotypes in automatic behaviour[END_REF] provide some insight. They suggested that although priming an outgroup can automatically activate the complementary ingroup (Wilder & Shapiro, 1984), this may only occur if the ingroup-outgroup dichotomy is salient. They argued that unless the perceiver interprets the prime as an outgroup stereotype rather than just a stereotype then contrastive responses may not be expected. These predictions were supported empirically;

increasing the salience of the outgroup designation of the target group led to a contrastive behavioral responses (Gordijn & Stapel, 2006;[START_REF] Schubert | Contrast from social stereotypes in automatic behaviour[END_REF]. Furthermore, Spears and colleagues (2004) have demonstrated that outgroup assimilation can be turned into outgroup contrast by making the intergroup context salient to perceivers. In sum, outgroup primes will

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lead to behavioral contrast, but only if an intergroup comparative context is salient; that is, only if they are thought about as outgroups by perceivers.

Ingroup identification as a moderator

It has been demonstrated that the salience of the intergroup context predicts whether assimilative or contrastive responses follow outgroup primes [START_REF] Brewer | Self-evaluation effects of interpersonal versus intergroup social comparison[END_REF]Gordijn & Stapel, 2006;[START_REF] Schubert | Contrast from social stereotypes in automatic behaviour[END_REF][START_REF] Spears | Reaction in action: Intergroup contrast in automatic behavior[END_REF], however all of these studies demonstrate that contextual manipulations of the salience of the intergroup context influence responses to outgroup primes. In the current research, we suggest that there may be a personcentered variable that may predict whether the intergroup context is spontaneously salient to perceivers. We argue that the perceiver's level of commitment to their ingroup will predict the tendency to contrast from an outgroup prime. This is because ingroup identification can be thought of as the extent to which the perceiver has an automatic tendency to self-define as an ingroup member. Empirically, the notion that commitment to ingroups is an important element in group perception and behavior is well supported. Highly identified perceivers are more likely to conform to group norms (Fielding & Hogg, 2000;Hogg, Turner, & Davison, 1990;Jetten et al., 1997;Jetten, Postmes, & McAucliffe, 2002;McAucliffe, Jetten, Hornsey, & Hogg, 2003;Schofield, Pattison, Hill, & Borland, 2003;Smith & Terry, 2003, Study 2;[START_REF] Terry | Attitude-behaviour relations: The role of in-group norms and mode of behavioural decision-making[END_REF], perceive the ingroup to be more homogenous (Castano & Yzerbyt, 1998 Study 2;DeCremer, 2001DeCremer, , 2003;;[START_REF] Doosje | Perceived intragroup variability as a function of group status and identification[END_REF]Ellemers, Spears, & Doosje, 1997;Lorenzi-Cioldi, Deaux, & Dafflon 1998;Jetten, Branscombe, Spears, & McKimmie, 2003 Study 1; [START_REF] Pickett | Assimilation and differentiation needs as motivational determinants of perceived in-group and out-group homogeneity[END_REF]Weenig, van der Salm, & Wilke, 2004) and show more overlap in the traits they assigned to the self and the ingroup than they did in traits assigned to the outgroup [START_REF] Riketta | Cognitive differentiation between self, ingroup, and outgroup: The roles of identification and perceived intergroup conflict[END_REF], see also [START_REF] Hogg | Intergroup relations and group solidarity: Effects of group identification and social beliefs on depersonalized attraction[END_REF]. In sum, higher identifiers are more likely to self-stereotype (Jetten et al., 2002).

If the ingroup category is central to a higher identifier's self-conception [START_REF] Henderson-King | Ingroup favouritism and perceived similarity: A look at Russian's perceptions in a post-Soviet era[END_REF]Jetten, et al., 1998) and level of commitment to the ingroup

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predicts how the perceiver responds to group based information, if follows that higher levels of ingroup identification may increase the probability of a perceiver encoding information in intergroup terms. Self-categorization theory argues that the level at which the perceiver selfcategorizes depends on the accessibility of the self-category (Bruner 1957 as cited in [START_REF] Turner | A self-categorization theory[END_REF]; "the degree of internalization of or identification with an ingroup-outgroup membership, the centrality and evaluative importance of a group membership in self-definition, is a major determinant of accessibility…" (Turner, 1987, p. 55). We would therefore argue that perceivers who strongly identify with a particular ingroup category will be more sensitive to perceiving possible intergroup comparisons, and will differentiate themselves from the outgroup by contrasting (self-stereotyping) responses accordingly. Building on the reasoning of [START_REF] Schubert | Contrast from social stereotypes in automatic behaviour[END_REF] and Spears and colleagues we hypothesize that outgroup contrast will be more likely to occur for higher identifiers because such perceivers are more likely to spontaneously perceive the situation in intergroup terms compared to lower identifiers. Put another way, higher identifying individuals will be people who are more likely (than lower identifiers) to perceive a group prime as an outgroup prime.

What about ingroup primes? Although previous research has demonstrated comparisons with ingroup standards leads to assimilative responses and outgroup targets lead to contrastive responses [START_REF] Mussweiler | I know you are, but what am I? Self evaluative consequences of judging the in-group and out-group members[END_REF], we do not expected that ingroup identification will moderate responses to ingroup primes. Thinking about ingroups does not imply an intergroup comparison, rather, it provides an inter-individual comparative context [START_REF] Turner | Rediscovering the social group: A self-categorisation theory[END_REF]. Therefore, while higher identifiers are expected to be more attuned to an intergroup context we do not expect them to assimilate more to an ingroup prime because there is nothing to suggest or invoke an ingroup-outgroup comparison.

Experiment 1

The aim of Experiment 1 was to provide an initial test of the hypothesis that ingroup identification will predict responses to outgroup but not ingroup primes. The intergroup context

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Outgroup Contrast 8 used was ingroup (young) versus outgroup (elderly). The experimental paradigm used was based on that employed by [START_REF] Bargh | Automaticity of social behavior: Direct effects of trait construct and stereotype activation on action[END_REF]. Participants were primed using a scrambled sentence task and subsequently their behavior was assessed. In this experiment, the behavior of interest was memory, because one of the features of the elderly stereotype is poor memory. It was expected that participants' primed with the outgroup would recall more words than those primed with the ingroup (see also [START_REF] Dijksterhuis | On the relation between associative strength and automatic behavior[END_REF]. This tendency to contrast from an outgroup stereotype (i.e., the elderly as forgetful) was expected to be amplified for higher versus lower identifiers As a subsidiary measure participants were also asked to list as many nightclubs as they could in the local city centre within a set time limit. It was expected that participants' primed with the outgroup would list more clubs than those primed with the ingroup with this tendency being amplified for higher identifiers

Method

Participants and design

Thirty-seven participants with a mean age of 19 years were randomly allocated to an ingroup (young) vs. outgroup (elderly) prime condition in a between-participants design. Ingroup identification was measured prior to the manipulation and treated as a continuous variable.

Procedure

Participants were informed that they would complete a number of tasks, and that the first of these was "a measure that we were attempting to validate for future use". The first measure was actually a four item measure of ingroup identification (adapted from Branscombe, Wann, [START_REF] Branscombe | In-group or out-group extremity: Importance of the threatened social identity[END_REF]Luhtanen & Crocker, 1992). Participants were asked to rate their agreement with the following items: "I identify strongly with other young people", "Being a young person is an important part of who I am", "I feel strong ties with other young people", and "I feel a sense of solidarity with other young people", on a scale anchored at 1 (Not at all) to 9 (Very much so). The four items formed a reliable scale (Cronbachs α = .61) 1 so were collapsed into a single measure by summing the scores.

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Outgroup Contrast 9

All participants were then asked to complete "a language comprehension task". This was a version of the Bargh, Chen and Burrows' (1996) sentence scrambling task. The elderly priming stimuli were taken directly from Bargh et al with one exception. The sentence that unscrambled to "Oranges are from Florida" was removed as this was intended to prime elderly but we did not believe our sample from the UK would associate Florida with elderly. In the young condition we used the same sentences that were the filler items in the elderly priming task and created the same number of new sentences to prime young as were used to prime elderly. The words used were; noisy, inconsiderate, loud music, impulsively, open-minded, carefree, nightclub, youth, independent, drinking, untidy, energetic, irresponsibly, rowdy, moody, ungrateful, lively, trendy, immature. All words were pre-tested with 20 participants and were found to be significantly more typical of young from the midpoint (5) of a 1 (Typical of young) to 9 (Typical of elderly) scale at p < .001 with the exception of independent where t (19) = -2.77, p = .012. Participants then completed the dependent measures as described below, then were thanked and debriefed.

Dependent Measures

Participants completed a lexical decision which consisted of 10 words; bulb, wood, essential, calendar, clock, ring, handle, picture, hotel and news and 10 non-words; twot, dode, koybeed, confarance, chiar, pust, filser, cotfer, ledhar, and raed. On completion of this task participants were asked to recall as many of the stimuli that were presented as they could. This was to ascertain whether the prime had affected the participants' memory as elderly people are on average more forgetful than younger people (Hess, 1994;Howard, 1996). Previous research has used a recall task to assess the effects of the elderly stereotype on automatic behavior [START_REF] Dijksterhuis | On the relation between associative strength and automatic behavior[END_REF].

Participants were also asked to list all the bars and clubs that they could think of in the city centre as a second measure of the accessibility of ingroup-related (self-stereotypic) thoughts as a subsidiary measure. On this measure we expected participants in the outgroup prime

Results and Discussion

Recall task

A moderated regression analysis revealed a main effect of prime, β = -2.95, t = -3.04, p = .005; overall participants assimilated to the outgroup prime more than the ingroup prime 2 . There was no main effect of ingroup identification, β = 0.10, t = 0.61, p = .55. The main effect of prime was qualified, however, by the predicted interaction between ingroup identification and prime condition, β = 2.90, t = 3.00, p = .005, see Figure 1.

---Insert Figure 1 about here ---

In the ingroup prime (young) condition simple slopes analysis revealed no significant relationship between ingroup identification and the number of words, β = -0.40, t = -1.87, p =

.08 (although we note the trend towards higher identification predicting recall of fewer words).

In the outgroup priming condition (elderly), the number of words recalled significantly increased as identification increased, β = 0.51, t = 2.32, p = .035. This supports the hypothesis that an outgroup prime would lead higher identifiers to show a larger contrastive effect than lower identifiers.

Listing task

Moderated regression on the number of clubs generated revealed a significant main effect of prime, β = -2.96, t = -3.39, p = .002. Those perceivers exposed to an outgroup prime (elderly) showed a contrast effect by listing (remembering) more clubs than those primed with the ingroup category (young).There was also a main effect of ingroup identification, β = 0.40, t = 2.81, p = .008, with the number of ingroup words listed increasing as ingroup identification increased. These effects were qualified by the predicted interaction between identification and prime β = 3.08, t = 3.53, p = .001, see Figure 2.

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Outgroup Contrast 11 ---Insert Figure 2 about here ---

In line with predictions simple slope analysis on the ingroup condition revealed that ingroup identification did not predict the number of clubs generated in the ingroup (young) condition, β = -0.12, t = -0.52, p = .61. Simple slope analysis on the outgroup (elderly) condition revealed that, in line with predictions, as ingroup identification increased the tendency to contrast also increased with more clubs being listed β = 0.76, t = 4.46, p < .001.

The findings from this experiment suggest that ingroup identification can predict responses to outgroup primes, with higher identifiers tending to contrast more than lower identifiers. In line with predictions ingroup identification did not predict responses to ingroup primes. 3 In Experiment 1 higher identifiers were more likely to have better memory when primed with the outgroup than lower identifiers. We argue that this represents behavioral contrast, but there is an alternative possibility. Perhaps higher identifiers are simply more alert in intergroup contexts. Put another way, if higher identifiers are particularly concerned with intergroup comparisons, then it stands to reason that when thinking about outgroups then they will pay more attention to the situation at hand, especially if it seems relevant to the comparison. It is possible that the higher identifiers were not showing better memory because they were behaviorally contrasting from the poor memory elderly stereotype, but perhaps they were simply paying more attention to the task, induced to do so by the intergroup comparison, and subsequently showed better memory. To rule out the possibility that it is simply the case that higher identifiers pay more attention and devote more cognitive resources to tasks at hand when aware of an intergroup comparative context we need a task that would indicate behavioral contrast from an outgroup but not increased attention. In other words, we needed a task on which higher identifiers would have to do worse than lower identifiers to indicate behavioral contrast. In Experiment 2 we tested our hypotheses with such a measure: Math performance in a gender comparative context.

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Experiment 2

In this experiment we focused on gender primes and math performance. Stereotypically females are expected to under perform on math tests compared to males (e.g., Nosek, Banaji, & Greenwald, 2002). This immediately brings to mind the literature on stereotype threat [START_REF] Steele | Stereotype threat and the intellectual test performance of African-Americans[END_REF] which has examined the impact of negative stereotypic expectancies on task performance. When a person's social identity (e.g., female) is attached to a negative stereotype, that person will tend to under perform in a manner consistent with the stereotype (poor at math) particularly when they are aware that they will be compared with an outgroup (male) who stereotypically perform well on the test dimension (see [START_REF] Aronson | When white men can't do math: Necessary and sufficient factors in stereotype threat[END_REF]; for similar findings with ethnicity and math performance). Recent findings have also shown that the stereotype threat effect may only inhibit women's math performance if gender is an important aspect of their self-definition [START_REF] Schmader | Gender identification moderates stereotype threat effects on women's math performance[END_REF] 4 .

Our aim here was to test if ingroup identification predicted contrast to outgroups in terms of gender primes and math performance -an effect, if observed, that could be seen as analogous to stereotype threat effects. However, it is important to note that stereotype threat does not require an outgroup comparison. In this literature typically perceivers are told that their responses will be compared to an outgroup who stereotypically perform well on the relevant dimension [START_REF] Aronson | When white men can't do math: Necessary and sufficient factors in stereotype threat[END_REF][START_REF] Schmader | Converging evidence that stereotype threat reduces working memory capacity[END_REF]Rosenthal & Crisp, 2006, Study 4;Spencer, Steele & Quinn, 1999, Study. 2). However, more subtle manipulations can also lead to the effect, such as having instructions read by a male (outgroup) experimenter [START_REF] Schmader | Gender identification moderates stereotype threat effects on women's math performance[END_REF], asking people to circle their group memberships on the front of the test book [START_REF] Steele | Stereotype threat and the intellectual test performance of African-Americans[END_REF] and simply telling the perceiver that the test is diagnostic of the ability in question [START_REF] Steele | Stereotype threat and the intellectual test performance of African-Americans[END_REF]. Furthermore, [START_REF] Shapiro | From stereotype threat to stereotype therats: Implications of a multi-threat framework for causes, moderators, mediators, consequences and interventions[END_REF] have recently argued that stereotype threat may not be a singular concept, but that there may be six core types of threat that fall under this umbrella term, differentiated by the combination of target of the threat (self-or ingroup) and the (perceived) source of the threat (self, ingroup others or outgroup

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Outgroup Contrast 13 others). The different manipulations described above could have effects on just one, a subset, or all of these core types of threat, which may or may not involve outgroup comparisons.

The point here is that while the basic conditions necessary for outgroup contrast (priming the outgroup) may also be sufficient conditions for stereotype threat (or may accentuate stereotype threat), they are not necessary conditions for stereotype threat (i.e., stereotype threat can occur without priming the outgroup). Thus, our focus on outgroup contrast here should be seen as complementary to, while at the same time distinct from, stereotype threat phenomena.

Based on our theoretical model and the findings from Experiment 1 we therefore predicted that following an outgroup prime higher identifiers would show behavioral contrast in the form of poorer performance on a math task compared to lower identifiers. As before, we did not expect ingroup identification to predict responses to ingroup primes.

Method

Participants and Design

Fifty-six female participants (mean age = 19 years) were randomly allocated to the ingroup (women) or outgroup (men) priming condition using a between-subjects design. Ingroup identification was measured prior to the priming manipulation.

Procedure

Participants were informed that we were collecting information for a series of pre-tests.

The first task participants completed was a measure of ingroup identification as used in Experiment 1. The four items formed a reliable scale (Cronbachs α = .82) so were collapsed into a single measure by summing the scores of the four items for each participant.

All participants then completed a priming manipulation adapted from Macrae, Milne, &

Stangor, (1994 see [START_REF] Dijksterhuis | Seeing one thing and doing another: Contrast effects in automatic behaviour[END_REF][START_REF] Haddock | Syrian science and smart supermodels: On the when and how of perception-behaviour effects[END_REF]. Participants were asked to spend five minutes writing a paragraph thinking about the typical behaviors, lifestyle and appearance attributes associated with the ingroup (women) or the outgroup (men)

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Outgroup Contrast 14 dependent on condition. Participants then completed the dependant measures as described below before being thanked and dismissed.

Dependant measures

Given that men are stereotypically expected to perform better on mathematics tests than women, we used a mathematics test as a behavioral measure of whether assimilation or contrast had occurred. There were two versions of the mathematics test used, both adapted from Bache (2005). The tests consisted of ten questions all at GCSE level (Secondary school examinations taken at age 16 in the UK). Examples include, "How many 6 ½ ounce glasses can be filled from a 32-ounce container of orange juice?" and "If $1 = £1.60, then $40 =?" Participants were given ten minutes to complete the as many of the question as they could.

Results and Discussion

Results were analyzed in terms of math accuracy. Tests of mathematics ability in the stereotype threat literature are often considered in terms of accuracy because it gives a better impression of participants' behavior when participants are unable to answer all questions in the time allowed (Inzlicht & Ben-Zeev, 2000;Johns, Schmader, & Martens, 2005;[START_REF] Schmader | Converging evidence that stereotype threat reduces working memory capacity[END_REF][START_REF] Schmader | The costs of accepting gender differences: The role of stereotype endorsement in women's experience in the math domain[END_REF][START_REF] Steele | Stereotype threat and the intellectual test performance of African-Americans[END_REF]. This was similarly the case with our participants; very few were able to complete all ten questions. An accuracy measure takes into account the number of questions attempted so in this context is arguably a more suitable measure. 5

Accuracy was calculated for each participant by summing number correct and dividing it by number attempted (and then multiplying by 100 to create a percentage). Moderated regression analysis on math accuracy revealed a main effect of prime, β = 2.41, t =2.59 p = .012, women primed with the outgroup (men) performed worse on the math task than women primed with the ingroup (women), a typical outgroup contrast effect. This effect was qualified, however, by the predicted significant interaction between prime and ingroup identification, β = -2.44, t =-2.65, p = .011, see Figure 3. Simple slope analysis revealed that ingroup identification did not

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predict math performance following an ingroup prime β = .26, t = 1.38, p = .181. Ingroup identification did however predict math performance following an outgroup prime β = -.39, t = -2.18, p = .039. Higher identifiers contrasted their responses by performing less accurately on the math task that lower identifiers.

---Insert Figure 3 about here ---These results provide further support for our predictions. Ingroup identification did not predict math performance in response to an ingroup prime but did in response to an outgroup prime. Higher identifiers showed an increased tendency to contrast their responses from outgroups. This later finding is in keeping with the stereotype threat literature, which has shown that women who know they are being compared with men perform worse on math tests.

Recently research by [START_REF] Schmader | Gender identification moderates stereotype threat effects on women's math performance[END_REF] has demonstrated that the tendency for women to perform worse on math tests following a threatening comparison may be predicted by ingroup identification. The current results echo Schmader's findings where a subtle activation of the outgroup (the presence of a male experimenter) led higher identifiers to perform worse on a math test (contrast).

The findings from this experiment support the hypothesis that higher identifiers will show contrastive responses and self-stereotype more when they have been exposed to an outgroup prime. The effect was observed regardless of the fact that contrast leads to poorer performance, ruling out the possibility that the effect is due simply to higher identifiers paying more attention in salient intergroup contexts. These findings suggest that the apparent desire for higher identifying participants to differentiate themselves from outgroups was so strong as to make these negative implications irrelevant in this context.

General Discussion

The experiments reported suggest that thinking about an outgroup may invoke comparison and contrast processes for those social perceivers who are highly identified with their ingroup, but not lower identifiers. It was predicted that the combination of higher ingroup

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identification and an outgroup prime would produce contrastive behavioral responses because such perceivers are attuned to the salience of the intergroup context and would want to define themselves as ingroup members by self-stereotyping. These two experiments provide consistent evidence that contrastive responses to outgroup primes can be predicted by perceivers' a priori level of commitment to the ingroup. This effect was replicated on different behavioral measures, in different intergroup settings, using different priming methodologies, suggesting that these findings are robust and generalizable. Importantly we also showed that the contrast effect was observed even when this would have negative implications for self-esteem (i.e., poorer performance). This removes the possibility that the observed effects are due to increased attention to relevant tasks in salient intergroup contexts, and supports the idea that behavioral contrast represent an identity motive to differentiate self-conceptually important ingroups from outgroups. There is however a limitation of the findings of the current experiments, which should be acknowledged before theoretical implications are discussed. In the current studies it is only possible to conclude that higher identifiers are contrasting more from an outgroup prime than lower identifiers, but one cannot dissociate whether the lower identifiers are assimilating more or contrasting less. This issue of relatively of findings is common in studies that investigate the effects of ingroup vs. outgroup primes. Future research would benefit from ingroups and outgroups being judged relative to a control condition to allow for a clearer specification of perceivers responses.

Theoretical implications

The research presented here is consistent with, and extends, previous work showing that when an outgroup is salient to perceivers contrast effects are likely to be observed [START_REF] Mussweiler | I know you are, but what am I? Self evaluative consequences of judging the in-group and out-group members[END_REF]Schubert & Häfner, 2002;[START_REF] Spears | Reaction in action: Intergroup contrast in automatic behavior[END_REF]. The current investigation refines these previous findings by demonstrating that highly identified ingroup members appear attuned to recognizing an outgroup as an outgroup. These studies provide clear evidence that There is considerable evidence that the extent to which the social perceiver identifies with their ingroup influences their responses to group-relevant stimuli. In this article we have argued that the perceiver's level of commitment to their ingroup predicts the tendency to contrast from outgroups. We suggest that the reason that higher identifiers self-stereotype or contrast more than lower identifiers is two fold. First, higher identifiers are more attuned to potential category comparisons [START_REF] Ellemers | Feedback and self-esteem: Individual and group level manipulations and responses[END_REF]. In the experimental paradigms used the intergroup context is not explicitly salient and for higher, but not lower, identifiers we observe effects consistent with an interpretation of a category as a comparative outgroup.

Second, higher identifiers, when aware of an intergroup comparison, will think and behave in terms of their ingroup identity, that is, self-stereotype and define themselves in terms of the ingroup norm (in comparison to lower identifiers).

More generally, the current investigation draws on research from the social identity perspective and the social comparison perspective. As Guimond (2006, p. 3) notes these "two major traditions of research both dealing with the social psychology of comparison, albeit each in distinctive ways, have ignored each other". The approach taken here has attempted to address both research domains and has provided insights for both research areas. First, for social comparison research, the findings suggest that individual differences and underlying motivations can influence our automatic behavior to social comparison information. Although the current research only addressed ingroup identification as an underlying drive to self-define as a group member, other researchers have also begun to consider individual differences (e.g. liking of the targets group, see Cesario, Higgins, & Plaks, 2006) and motivational moderators (e.g. selfprotective motivations, see [START_REF] Stapel | When nothing compares to me: How defensive motivations and similarity shape social comparison effects[END_REF][START_REF] Stapel | Defensive social comparisons and the constraints of reality[END_REF] of responses to social comparison information. Second, these studies have provided insight for social identity theory in that they provide evidence that ingroup identification is an important

A C C E P T E D M A N U S C R I P T Outgroup Contrast 18
moderator of implicit responses to group stimuli. This is a finding currently echoed in the literature on reducing prejudice and intergroup conflict where high ingroup identification has been shown to inhibit the effectiveness of attempts at bias reduction, even at an implicit level [START_REF] Crisp | Recategorization and subgroup identification: Predicting and preventing threats from common ingroups[END_REF][START_REF] A C C E P T E D M A N U S C R I P T Outgroup Contrast 20 Crisp | Reducing implicit prejudice by blurring intergroup boundaries[END_REF]. Together these implications support the idea that considering social comparison and social identity approaches alongside one another can be a fruitful endeavor.

Conclusions

The research reported in this article demonstrates that perceivers' underlying commitment to the groups to which they belong may not only alter their strategic responses but also more implicit responses. Higher ingroup identification is associated with an increased tendency to contrast from an outgroup prime. We suggest that this tendency stems from an increased tendency to self-categorize as an ingroup member and define the self in ingroup terms.

More broadly, these findings suggest that while perceivers' behavior may be shaped by stimuli they are unaware of, this influence may be moderated by their own motivations. The findings we report suggest that that the link between perception and automatic behavior is not a simple one, and that unconscious desires and motivations may intervene between perception and action. Footnotes 1 A higher Cronbachs α (α= .691) was achieved when the second item, "being a young person is an important part of who I am" was removed from the four item scale. An analysis of the three item scale showed the same pattern of results as the four-item measure for both recall and the listing task, consequently for continuity and clarity we present the four-item measure as it was also used in Experiment 2.

A C C E P T E D M A N U S C R I P T

2 There was a significant outgroup assimilation effect of prime on the recall task. Examining the interaction it seems that this main effect is driven by lower identifiers remembering fewer words, i.e., assimilation towards the outgroup stereotype (elderly). This is generally consistent with our theoretical model: We argue that higher identifiers will show a more pronounced contrast from outgroups and therefore by implication lower identifiers will be more likely to assimilate.

3 In Experiment 1 ingroup identification was measured directly before the group priming manipulation. As such it is possible that it was the short interval between the identity measure and the prime that was responsible for these effects. A pilot study was conducted to address this issue by measuring perceivers' ingroup identification 24 hours before the priming task and the behavioral measure. The results revealed the same pattern of responses as observed in Experiment 1. A moderated regression analysis on time taken to complete the task revealed a significant main effect of priming condition, β = 1.90, t = 2.14, p = .043 such that those primed with the outgroup contrasted to the outgroup prime. Those perceivers exposed to an outgroup prime (elderly) took less time than those primed with a neutral prime: An outgroup contrast effect. The predicted interaction between priming condition and ingroup identification was significant, β = -2.07, t = -2.34, p = .027.

A C C E P T E D M A N U S C R I P T

Outgroup Contrast 32 in the test domain) with higher identifying females. Here we compare higher identifying females simply primed with the outgroup to those primed with the ingroup.

5 Although we advocate math accuracy as our performance measure we also analyzed the total number of questions answered correctly and note that the same pattern of results was obtained (prime x ingroup identification interaction, β = -1.86, t =-1.98, p = .05).

  more bars and clubs. Participants were given two minutes to complete this task.

  commitment and desire to self-define as an ingroup member influences their responses to outgroup primes.

Figure 1 -

 1 Figure 1-Word recall as a function of priming condition and ingroup identification (Exp 1).

Figure 2 -

 2 Figure 2-Clubs listed as a function of priming condition and ingroup identification (Exp 1).

Figure 3 -

 3 Figure 3-Math accuracy as a function of priming condition and ingroup identification (Exp 2).

This experiment is different to[START_REF] Schmader | Gender identification moderates stereotype threat effects on women's math performance[END_REF] study in several ways. She used a different threat manipulation and compared higher identifying males (who may exhibit stereotype lift, an increase in performance due to the association of the perceiver's group with a positive stereotype

This research was funded in part by an Economic and Social Research Council doctoral studentship (PTA-030-2002-00889) to N. R. Hall.


Alina Hedrych-Ozimina

Kristina Behrendt

Zhenyue Hao

Ruth Pofahl

Doreen Ussath

Renate Knaup

Thomas Krieg

Ingo Haase
email: ingo.haase@uni-koeln.de

Enhanced contact allergen and UVB induced keratinocyte apoptosis in the absence of CD95/Fas/Apo-1

Keywords: CD95, Fas, epidermis, apoptosis, sun burn cell, eczematous dermatitis

FAS/ CD95/ Apo-1 is an ubiquitously expressed cell surface receptor involved in the initiation of programmed cell death. Its function in epidermal keratinocytes has been incompletely defined. Available evidence from in vitro studies points to important roles of Fas in the pathogenesis of contact dermatitis and in keratinocyte apoptosis induced by ultraviolet light. To define functions of Fas in the epidermis in vivo, we have generated mice with epidermis specific deletion of the fas gene and have tested its requirement for DNFB induced contact dermatitis and for UVB induced keratinocyte apoptosis. We report here our unexpected finding that keratinocyte apoptosis induced by both a contact allergen and UVB irradiation was significantly enhanced in Fas negative epidermis. Expression of Fas by epidermal keratinocytes was neither necessary for the normal development of contact hypersensitivity of the skin, nor required for keratinocyte apoptosis following UVB irradiation. Our results thus demonstrate that, in the epidermis in vivo, Fas exerts anti apoptotic functions that outweigh its pro apoptotic role in contact hypersensitivity responses of the skin and in the tissue response of the epidermis to UVB irradiation.

Introduction

Fas (APO-1/CD 95) is a widely expressed cell surface receptor belonging to the TNF receptor superfamily. The receptor chains are expressed on the cell surface as pre associated homotrimers. Binding of its cognate ligand, FasL, is thought to result in super clustering to form oligomeric receptor complexes. As a consequence, several proteins accumulate next to the cytoplasmic domain of Fas and form the death inducing signaling complex (DISC) [START_REF] Peter | The CD95(APO-1/Fas) DISC and beyond[END_REF].

Two important components of the DISC are the adaptor protein FADD and its binding partner caspase-8. Caspase-8 is a cysteine protease functioning as initiator of a cascade of proteolytic cleavage steps that finally leads to the activation of effector caspases. Effector caspases, like the cystein protease caspase-3, cleave various intracellular proteins thereby inducing cellular changes that cause the phenomenon of apoptosis. In cells, caspase-3 is sequestered as a zymogen (p32 caspase-3). Upon activation of the pro apoptotic effector cascade this is converted into active caspase-3 by proteolytic cleavage. Activation of caspase-3 can be detected in tissues and cells by immunostaining using cleavage site specific antibodies [START_REF] Gown | Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3[END_REF].

Epidermal keratinocytes have been shown to express Fas (3) and ligation of Fas with a specific antibody can induce apoptosis of keratinocytes in culture after preincubation with gamma interferon. In addition, supernatant from activated human T-lymphocytes containing both FasL and gamma interferon induced apoptosis of cultured human keratinocytes.

Apoptosis of Fas-expressing epidermal keratinocytes in association with T-lymphocytes expressing FasL has also been described in contact dermatitis and atopic dermatitis and were thought to play an important role in the pathogenesis of these conditions [START_REF] Trautmann | T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis[END_REF]. The dissociation of epidermal keratinocytes with breakup of cell-cell contacts and formation of intraepidermal blisters, a phenomenon called spongiosis, has been thought to be mediated by Fas/ FasL-interactions. In addition to Fas/ FasL-interactions, perforin expressed by cytotoxic T-lymphocytes can cause killing of keratinocytes. This additional mechanism of killing was shown to be of relevance in contact dermatitis in mice [START_REF] Kehren | Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity[END_REF][START_REF] Kagi | Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity[END_REF].

In addition to its role in initiating apoptosis, Fas-signaling has also been proposed to activate non cell autonomous anti-apoptotic mechanisms in eczematous dermatitis. Upon induction of Fas-signaling, epidermal keratinocytes produce ligands for the epidermal growth factor receptor (EGFR) such as amphiregulin, transforming growth factor alpha (TGF-α) and others. These have been shown to activate anti apoptotic EGFR-signaling in neighboring keratinocytes to protect them from apoptosis and thus restrict tissue damage. In addition, EGFR-dependent signals induced by Fas stimulation in keratinocytes have been implicated in the production of pro inflammatory cytokines that could contribute to the inflammatory reaction in eczematous dermatitis [START_REF] Iordanov | Cell death-induced activation of epidermal growth factor receptor in keratinocytes: implications for restricting epidermal damage in dermatitis[END_REF][START_REF] Farley | Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases[END_REF].

Keratinocyte apoptosis in skin exposed to ultraviolet light (UV light) is characterized by the formation of sunburn cells (SBC). This is thought to be the main mechanism by which potentially transforming mutations are eliminated from the skin. UV light induces DNA damage which leads, in case of irreparability, to p53-mediated apoptosis of the damaged cell [START_REF] Ziegler | Sunburn and p53 in the onset of skin cancer[END_REF]. The activity of DNA repair mechanisms, which determines whether keratinocytes undergo apoptosis or not, can be regulated by factors of the extracellular milieu [START_REF] Schwarz | Interleukin-12 suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair[END_REF]. In addition to this DNA damage response, apoptosis of epidermal keratinocytes induced by UV light is regulated by Fas dependent mechanisms [START_REF] Kulms | Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation[END_REF]. Irradiation of human epidermal keratinocytes with UVB induces clustering of Fas on the cell surface in a ligand independent manner and recruitment of FADD to the cytoplasmic domain of Fas. This is thought to trigger the same caspase-8 dependent pro apoptotic signaling cascade that is also induced after binding of FasL to Fas and leads to the activation of Caspase-3 [START_REF] Kulms | Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation[END_REF][START_REF] Aragane | Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L[END_REF]. Similar results have been described for murine epidermal keratinocytes [START_REF] Takahashi | Ultraviolet B irradiation induces apoptosis of keratinocytes by direct activation of Fas antigen[END_REF][START_REF] Takahashi | Fas antigen modulates ultraviolet B-induced apoptosis of SVHK cells: sequential activation of caspases 8, 3, and 1 in the apoptotic process[END_REF]. In line with this proposed mechanism, studies in mice with mutated FasL suggested that up regulation of Fas and FasLexpression after irradiation with high doses of UVB contribute to apoptosis of epidermal keratinocytes [START_REF] Hill | Fas ligand: a sensor for DNA damage critical in skin cancer etiology[END_REF]. However, UVB induced keratinocyte apoptosis has also been described to proceed via the internal, mitochondrial pathway. UV light is thought to damage the mitochondrial membrane thus leading to the activation of caspase-9, a critical step in the initiation of apoptosis via this pathway. Blocking caspase-9 as well as targeted deletion of JNK can inhibit UV-induced apoptosis [START_REF] Sitailo | Activation of caspase-9 is required for UVinduced apoptosis of human keratinocytes[END_REF][START_REF] Tournier | Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway[END_REF], while blocking of caspase-8 did not prevent UV-dependent apoptosis in murine embryonic fibroblasts (MEFs) [START_REF] Naik | Ultraviolet radiation triggers apoptosis of fibroblasts and skin keratinocytes mainly via the BH3-only protein Noxa[END_REF][START_REF] Varfolomeev | Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally[END_REF]. This suggested that caspase-8 mediated Fas signaling may not be essential for the proapoptotic response upon UVB-irradiation in MEFs.

Given partially contradictory results regarding functions of Fas in keratinocytes we decided to use conditional gene targeting technology to further elucidate the role of Fas in the epidermis in vivo.

Results

Generation of epidermis specific Fas knockout mice

Mice with conditional ablation of Fas alleles have been described previously [START_REF] Hao | T cell-specific ablation of Fas leads to Fas ligand-mediated lymphocyte depletion and inflammatory pulmonary fibrosis[END_REF]. Female mice homozygous or heterozygous for the floxed Fas allele were bred to male mice homozygous or heterozygous for the floxed Fas allele and expressing Cre-recombinase under the control of the keratin 14-promoter [START_REF] Hafner | Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyteexpressed protein[END_REF]. Samples of epidermis from ear and back skin as well as total tissue lysates from mouse tails for comparison were analysed for the presence of wild type and mutant Fas-DNA (supplem. Fig. 1a) and of Fas-protein using PCR and Western blotting, respectively. Whereas epidermal samples from ear and back skin showed complete deletion of floxed Fas-alleles, Fas was readily detected in total tissue lysates from tails (Fig. 1a, supplem. Fig. 1b). This demonstrated epidermis specific deletion of Fas in mice with the genotype K14Cre + Fas fl/fl which will be referred to as Fas E-KO mice. Mice with the genotypes K14Cre -Fas fl/fl , K14Cre -Fas fl/+ , K14Cre + Fas +/+ and K14Cre -Fas +/+ are referred to as control mice. Mice with the genotype K14Cre + Fas fl/+ were excluded from the analysis.

We also analysed primary keratinocytes isolated from control and Fas E-KO mice. Western blot and FACS-analysis showed expression of Fas by keratinocytes from control mice. This was not detectable in keratinocytes isolated from Fas E-KO mice (Fig. 1 b,c). In addition, we tested the response of interferon gamma treated control and Fas KO keratinocytes to stimulation of Fas using the agonistic antibody Jo2 [START_REF] Ogasawara | Lethal effect of the anti-Fas antibody in mice[END_REF]. Whereas control keratinocytes showed an increase in the percentage of apoptotic cells upon Jo2 stimulation, the number of apoptotic cells was not increased in the Fas KO keratinocyte population (supplem. Fig. 2a).

Fas E-KO mice did not show an overt phenotype (supplem. Fig. 2b). Their skin was macroscopically and histologically indistinguishable from that of control mice. We also did not observe differences in the hair coat. Staining for markers of proliferation and differentiation in the skin of Fas E-KO mice and control mice did not show any differences (suppl. Fig. 3).

Enhanced contact dermatitis induced keratinocyte apoptosis in Fas E-KO mice

Fas has been proposed to exert an important function in the pathogenesis of contact dermatitis by mediating apoptosis of epidermal keratinocytes (4). We induced a contact hypersensitivity (CH) reaction in the ears of 16 Fas E-KO mice (9 females and 7 males) and 14 control mice ( 6females and 8 males) by challenging with 2, 4-di-nitrofluorobenzene (DNFB) in acetone/olive oil 4:1, 6 days after sensitization. Ear swelling was determined at different time points after challenge as a marker for the strength of the inflammatory reaction. Ear thickness was determined on the day of sensitization and immediately prior to challenge. The average of these measurements formed the baseline value for the determination of ear swelling.

Measurements of ear thickness were repeated 24 h, 48 h, 72 h and 96 h after challenge. No significant differences were detected between Fas E-KO mice and control mice at any time point (Fig. 2 a). Females showed stronger ear swelling than males through all time points. Both Fas E-KO mice and control mice showed edema and an inflammatory infiltrate in the dermis with similar density and comparable numbers of inflammatory cells staining positive for CD3 (T-lymphocytes) or CD11b (myeloid inflammatory cells) (Fig. 2 b). Similar numbers of F4/80 positive inflammatory cells (macrophages) were detected in the epidermis at sites of spongiosis both in control and Fas E-KO mice.

Spongiosis was present in all skin samples of challenged Fas E-KO mice, to a similar extent as in control mice (Fig. 2 b). We used immunostaining of activated caspase-3 and TUNELstaining (Fig. 3 a) to visualize intra epidermal apoptotic cells in challenged ears of Fas E-KO mice and control mice 96 hours after challenge. Unexpectedly, the number of apoptotic cells in the epidermis was higher in both female and male Fas E-KO mice as compared to controls.

The differences between Fas E-KO mice and control mice were significant (Fig. 3 b). We conclude that, in the DNFB-induced CH reaction in mice, 1) Fas expressed by epidermal keratinocytes is dispensable for the ear swelling reaction, the accumulation of an inflammatory infiltrate and spongiosis, and 2) the presence of Fas in the epidermis partially protects epidermal keratinocytes from apoptosis.

Enhanced UVB induced keratinocyte apoptosis in Fas E-KO mice

Fas has also been implicated in the apoptosis of keratinocytes following UVB-irradiation. We therefore irradiated 15 Fas E-KO mice and 13 age matched control mice with a dose of 1 J/cm 2 UVB on their back after shaving. After 18 and 24 hours, back skin from 8 male Fas E-KO mice and 9 male control mice or 7 female Fas E-KO mice and 4 female controls, respectively, was taken for analysis. Histological sections of paraffin embedded skin were used for counting sun burn cells and for TUNEL-staining. While there was no difference in the number of TUNELpositive cells in the epidermis of control und Fas E-KO mice, there were significantly more sun burn cells present in the skin of Fas E-KO mice Fig. 4 b). Staining with an antibody against the macrophage marker F4/80 did not show positive cells within the epidermis. We conclude that expression of Fas by epidermal keratinocytes is not required for UVB-induced keratinocyte apoptosis in vivo and that the presence of Fas reduces the number of sunburn cells upon UVB-irradiation.

No requirement for Fas in UVB induced keratinocyte apoptosis in vitro

In vivo, there is constant exchange of information between the epidermis and other skin compartments. To investigate whether deletion of Fas had an influence on cell autonomous mechanisms of keratinocyte apoptosis we isolated keratinocytes from Fas-negative epidermis and control epidermis. Western blot and FACS-analysis showed that keratinocytes isolated from Fas E-KO mice (Fas KO keratinocytes) did not express Fas protein (Fig. 1b,c). Control and Fas KO keratinocytes, 3 different lines each, were then irradiated in vitro with 100 and 300 mJ/cm 2 UVB and analyzed by FACS using an antibody against annexin V and by TUNELstaining. Both Annexin V and TUNEL-staining showed similar numbers of apoptotic control and Fas KO keratinocytes (Fig. 5). We conclude that apoptosis of keratinocytes upon UVBirradiation does not depend on the presence of Fas.

Discussion

Epidermal keratinocytes constitutively express CD95 (3) and Fas induced keratinocyte death is currently thought to play a relevant role in the pathogenesis of a number of severe skin diseases such as eczema and toxic epidermal necrolysis [START_REF] Trautmann | T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis[END_REF][START_REF] Borchers | Stevens-Johnson syndrome and toxic epidermal necrolysis[END_REF]. Although apoptosis of epidermal keratinocytes is considered as a hallmark of eczematous dermatitis [START_REF] Schwarz | No eczema without keratinocyte death[END_REF] the biological meaning of this is not well understood. The results from experimental models of contact dermatitis are inconclusive: The absence of Fas or FasL in mice has been reported to have no influence on the contact hypersensitivity response to DNFB [START_REF] Kehren | Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity[END_REF]. In another study, however, oxazolone-induced contact dermatitis was found to be attenuated in Fas-mutant lpr mice [START_REF] Xu | Role of Fas/Fas ligand-mediated apoptosis in murine contact hypersensitivity[END_REF]. Since both studies were performed in animals with constitutive functional Fas or FasL deficiency, they were not able to specifically address contributions of epidermal keratinocytes to the tissue reaction. Our approach of an epidermis specific deletion of Fas clearly shows that Fas expression by epidermal keratinocytes is dispensable for a normal contact hypersensitivity response, as ear swelling and inflammatory features were identical in control and Fas deficient skin. The formation of spongiotic vesicles, which is seen as a characteristic sign of acute eczematous dermatitis and has been proposed to be induced by Fas-mediated keratinocyte killing (4), occurred normally in the absence of epidermal Fas expression, showing that it is not Fas dependent. It is likely that, in the absence of Fas, keratinocyte killing is executed by perforin/ granzyme dependent mechanisms [START_REF] Kehren | Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity[END_REF].

In addition to its role in the induction of apoptosis, Fas has been suggested to elicit antiapoptotic and pro-inflammatory signals in the epidermis through the production of EGFreceptor ligands such as amphiregulin, TGF-α and numerous pro inflammatory cytokines [START_REF] Iordanov | Cell death-induced activation of epidermal growth factor receptor in keratinocytes: implications for restricting epidermal damage in dermatitis[END_REF][START_REF] Farley | Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases[END_REF][START_REF] Farley | Fas ligand elicits a caspase-independent proinflammatory response in human keratinocytes: implications for dermatitis[END_REF]. Recent work shows that the intracellular domain of Fas can be tyrosine phosphorylated, which can lead to the recruitment and activation of phosphatidylinositol-3kinase (PI-3K) [START_REF] Martin-Villalba | Tyrosine phosphorylation and CD95: a FAScinating switch[END_REF]. These findings are based on in vitro studies of immortalized and primary human keratinocytes and other cell types; their relevance for the in vivo situation within the epidermis remained therefore unclear. Our unexpected result showing that keratinocyte apoptosis upon DNFB-challenge is enhanced in Fas negative epidermis demonstrates an anti apoptotic function of Fas and it is possible that EGF-receptor ligands or PI-3K signaling mediate this protective signal. This mechanism could indeed restrict extensive tissue damage in response to pro apoptotic stimuli thus contributing to the maintenance of an intact skin barrier [START_REF] Iordanov | Cell death-induced activation of epidermal growth factor receptor in keratinocytes: implications for restricting epidermal damage in dermatitis[END_REF]. Probably, this function of Fas is mediated in a non cell autonomous juxtacrine way, since we did not detect the protective effect against apoptosis in cultures of Fas KO keratinocytes. Although this is, in our view, the most likely mechanism explaining the accumulation of apoptotic cells in the epidermis of Fas E-KO mice, we cannot completely exclude the possibility that the deficiency of Fas in epidermal keratinocytes leads to an inhibition of their autophagocytic activity. This would result in a decreased elimination of apoptotic cells and thereby in their accumulation. Such a function for Fas has, however, so far not been reported. In contrast, the death effector domain containing cellular FLICE-like inhibitor protein c-FLIP which can be recruited to the death inducing signaling complex (DISC) via the adapter protein FADD upon Fas-ligation, has been shown to suppress autophagy [START_REF] Lee | FLIP-mediated autophagy regulation in cell death control[END_REF].

Stimulation of Fas in epidermal keratinocytes has also been reported to result in the production of pro inflammatory cytokines that could facilitate the inflammatory response in eczematous dermatitis [START_REF] Farley | Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases[END_REF][START_REF] Farley | Fas ligand elicits a caspase-independent proinflammatory response in human keratinocytes: implications for dermatitis[END_REF]. On the basis of these findings, a possible pathogenic role of Fas-induced cytokine production in eczema has been assumed [START_REF] Leverkus | CD95-mediated signals in the skin: going out with an (inflammatory) bang?[END_REF]. We show here that epidermis specific deficiency of Fas does not alter the contact hypersensitivity response to a DNFB-challenge in mice. While our results do not exclude production of pro inflammatory cytokines in the epidermis upon Fas ligation they show that the presence of Fas is not required for the pathogenesis of contact dermatitis in vivo.

Keratinocyte apoptosis upon UV irradiation and subsequent elimination of apoptotic bodies

from the skin is believed to be the mechanism that protects the epidermis from cumulating DNA-damage in sun exposed skin. UV-light is absorbed by nuclear, cytoplasmic or cell membrane attached molecules that serve as chromophores. Excitation of these chromophores can activate a number of signaling pathways leading to apoptosis. Fas has been shown to cluster on the surface of human epidermal keratinocytes upon UVB-irradiation in a FasLindependent manner. This is followed by the recruitment of the adapter molecule FADD to the receptor complex which then enables the recruitment of caspase-8. Both a dominant negative FADD construct and inhibition of caspase-8 could reduce UVB induced apoptosis suggesting a causal role of the Fas-FADD-caspase 8 interaction in the induction of apoptosis [START_REF] Kulms | Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation[END_REF][START_REF] Aragane | Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L[END_REF][START_REF] Rehemtulla | Ultraviolet radiation-induced apoptosis is mediated by activation of CD-95 (Fas/APO-1)[END_REF]. This role of Fas was further supported by the finding that 500 mJ/cm 2 UVB irradiation of C3H/HeJ mice with mutated FasL (gld/gld mice) resulted in less apoptosis as compared to wild type controls [START_REF] Hill | Fas ligand: a sensor for DNA damage critical in skin cancer etiology[END_REF]. In contrast to these findings, we show here that in C57/Bl6 mice the Fas/ FasL-signaling system is not essentially involved in UVB-induced keratinocyte apoptosis. Both counting of sun burn cells and TUNEL-staining revealed that there is no decrease in the numbers of apoptotic keratinocytes in Fas negative epidermis as compared to control epidermis. This is further supported by our in vitro analysis of UVBirradiated Fas KO and control keratinocytes which did not show differences in Annexin Vand TUNEL-staining. These results are consistent with data showing that deletion or blockade of caspase-8 does not prevent UVB-induced apoptosis of MEFs [START_REF] Naik | Ultraviolet radiation triggers apoptosis of fibroblasts and skin keratinocytes mainly via the BH3-only protein Noxa[END_REF][START_REF] Varfolomeev | Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally[END_REF].

Furthermore, the increased number of sunburn cells in the epidermis of Fas E-KO mice suggests that, in vivo, Fas exerts anti apoptotic functions. TUNEL staining did not show a difference in the numbers of apoptotic keratinocytes between Fas E-KO mice and control mice. This is most likely due to the relatively late time point of 18 hours post irradiation, which was chosen primarily for the detection of sunburn cells.

The difference between our results and results from other models of UVB-induced keratinocyte apoptosis may be explained by variations in the UV response between mouse strains. Another explanation could be a gradual involvement of different pro apoptotic mechanisms depending on the UVB-dose, since Hill et al. [START_REF] Hill | Fas ligand: a sensor for DNA damage critical in skin cancer etiology[END_REF] used 500 mJ/cm 2 whereas we used 300 mJ/cm 2 . A third hypothetical explanation is offered by the fact that gld/gld mice are not deficient for FasL but carry a point mutation within the protein that disables pro apoptotic Fas-signaling [START_REF] Takahashi | Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand[END_REF]. As a consequence, mutant FasL-mRNA is constitutively and strongly expressed in lymphoid cells of gld/gld mice [START_REF] Watanabe | Constitutive activation of the Fas ligand gene in mouse lymphoproliferative disorders[END_REF]. It is therefore conceivable that mutant FasL in these mice, although not able to stimulate pro apoptotic Fas signaling, can still bind to CD 95 expressed on the surface of epidermal keratinocytes and activate its anti apoptotic signaling mechanisms. This would then lead to the stimulation of survival signals in keratinocytes and thus result in decreased apoptosis. The ability of Fas to stimulate pathways involved in cell survival has been demonstrated recently [START_REF] Kleber | Yes and PI3K bind CD95 to signal invasion of glioblastoma[END_REF]. In addition, signaling via c-FLIP, which is known to exert strong anti-apoptotic effects by binding to the adapter protein FADD and competing for the recruitment of caspase 8, could be a mechanism mediating anti apoptotic activities of Fas [START_REF] Bagnoli | Cellular FLICE-inhibitory protein (c-FLIP) signalling: a key regulator of receptor-mediated apoptosis in physiologic context and in cancer[END_REF].

In summary, we here provide genetic evidence showing conclusively that, in murine skin, Fas is an essential element of pathways that restrict keratinocyte apoptosis in eczematous dermatitis and upon UVB-irradiation. Neither contact allergen nor UVB-induced keratinocyte apoptosis depend on the expression of Fas by epidermal keratinocytes in vivo.

Moreover, both the inflammatory tissue reaction and spongiosis in acute eczematous dermatitis can proceed in the absence of epidermal Fas-expression. The exact mechanisms of anti apoptotic Fas-signaling remain unclear here; both Fas-mediated autocrine stimulation of EGF-receptor dependent signals [START_REF] Iordanov | Cell death-induced activation of epidermal growth factor receptor in keratinocytes: implications for restricting epidermal damage in dermatitis[END_REF] and/ or activation of phosphatidylinositol-3-kinase [START_REF] Kleber | Yes and PI3K bind CD95 to signal invasion of glioblastoma[END_REF] could be relevant in this context. Both in contact dermatitis and upon UVB-irradiation the protective effects of Fas apparently outweigh Fas-induced keratinocyte death. It therefore seems possible that, for the maintenance of tissue integrity within the epidermis, anti apoptotic functions of Fas are at least as important as its pro apoptotic functions.

*Materials and Methods

Generation of mice with epidermis specific Fas deficiency and UV irradiation

Mice with conditionally targeted Fas-alleles have been described previously [START_REF] Hao | T cell-specific ablation of Fas leads to Fas ligand-mediated lymphocyte depletion and inflammatory pulmonary fibrosis[END_REF]. These mice were crossed with mice expressing Cre-recombinase under the control of the keratin-14 promoter [START_REF] Hafner | Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyteexpressed protein[END_REF]. Deletion of floxed Fas-alleles and presence of the Cre-recombinase transgene were analysed by PCR as described therein. For UVB-treatment mice were anaesthetized, shaved on their backs and irradiated with a dose of 300 mJ/cm 2 UVB using a Waldmann UV 801 lamp equipped with UV21 bulbs emitting UV-light in the range of 280-360 nm with an emission maximum at about 314 nm (Waldmann, Villingen-Schwenningen, BRD). The UVdose was determined using a Variocontrol UV-radiometer (Waldmann). Mice were killed after 18 or 24 hours and skin was taken for analysis.

Western blot analysis of epidermis and cultured keratinocytes

Epidermis was separated from dermis with forceps after 30 minutes of incubation in 3.8% ammonium thiocyanate dissolved in borate buffer pH 7.6 at 4 °C. Epidermis samples were minced and homogenized for 2 minutes on ice using an MM300 ultrasound tissue homogenizer (Retsch GmbH, Haan, Germany), extracted for 1 h in modified RIPA-buffer containing 5 mM EDTA, 1% Triton X 100, 1% NP40, 0,1% SDS, 0,5% deoxycholate, 20 µM leupeptin, 1 mM PMSF, 0.5 mg/ml soybean trypsin inhibitor, 0.5 mM NaVO 3 and 10 mg/ml p-nitrophenylphosphate, and centrifuged at 14,000 x g for 10 minutes at 4 °C. The supernatant was used for protein analysis. Keratinocytes were lysed in situ using the same buffer, scraped from the dishes and sonicated for 30 seconds at full power. Lysates were centrifuged at 14,000 x g for 10 minutes and the supernatant was used for Western blot analysis. 30 µg of protein were separated by SDS-PAGE and blotted onto nitrocellulose LC 2000 membranes (Invitrogen, Carlsbad, USA). After blocking with 5% milk powder solution the membrane was probed with the following antibodies: rabbit polyclonal anti Fas M-20 (Santa Cruz Biotechnology, USA) and mouse monoclonal anti actin antibody (MP Biomedicals, Aurora, USA). Immunoreactive proteins were visualised with HRP coupled secondary antibodies on Hyperfilm by chemiluminescence (Western Lightning, Perkin Elmer, Rodgau, USA). Protein quantification was carried out using the Pierce protein quantification kit (Pierce).

Induction of contact dermatitis

Mice were anaesthetized on day 0 and the thickness of the left ear was measured twice. Mice were shaved on the abdomen and 25 μl of a 0,5% DNFB-solution in acetone/olive oil 4:1 was applied and allowed to dry. Six days later the mice were again anaesthetized and the thickness of the left ear was determined twice. The mean of thickness measurements on day 0 and day 6 formed the zero value for the stimulation experiment. 10 µl of a 0.2% DNFB solution were applied to the front side and back side of the left ear. On days 7 -10 after sensitization mice were anaesthetized and the thickness of the left ear was determined twice. The relative increase in ear thickness was calculated for each time point.

Histopathological analysis and immunostaining

After excision, tissue samples were fixed in 4% paraformaldehyde or embedded in OCTcompound and frozen immediately. Further processing, paraffin embedding of paraformaldehyde fixed tissue and H/E staining was according to standard histopathological procedures. Immunostaining of keratin-14, keratin-10 and loricrin were performed as described in [START_REF] Stratis | Localized inflammatory skin disease following inducible ablation of I kappa B kinase 2 in murine epidermis[END_REF]. For immunostaining against activated caspase-3 on paraffin embedded tissue a rabbit antibody against cleaved caspase-3 (Cell Signaling Technology, Inc., Denver, USA) at a dilution of 1:50 was used according to the instructions of the manufacturer.

TUNEL-staining

TUNEL-staining of tissue sections and cells was carried out with the Dead End Fluorimetric TUNEL-System (Promega Corporation, Madison, USA) according to the instructions of the manufacturer.

Isolation and culture of epidermal keratinocytes, UV irradiation and Fas stimulation

Epidermal keratinocytes were isolated from transgenic and wild type newborn mice as described in [START_REF] Tscharntke | Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1[END_REF]. For UVB-irradiation, keratinocytes were grown to subconfluency without feeder cells, washed with PBS and then irradiated with 100 or 300 mJ/cm 2 UVB using a Bio Sun++ System at 312 nm (Vilber Lourmat, Marne-la-Vallée, France). After 16 hours of culture keratinocytes were trypsinized and analysed by FACS or TUNEL-staining. For stimulation of Fas, primary epidermal keratinocytes were incubated with 10 ng/ml IFNγ (kindly provided by Jonathan Howard, University of Cologne) to increase Fas-expression.

After 48 hours cells were stimulated with 50 ng/ml Jo2 antibody (Becton Dickinson #554256) or left unstimulated. 8 hours or 20 hours later keratinocytes were fixed and numbers of TUNEL-positive cells were determined.

FACS-analysis

Keratinocytes were trypsinized and resuspended in HEPES incubation buffer. Staining of Annexin-V was performed using the Annexin-V-FLUOS Staining Kit (Roche Applied Science, Penzberg, Germany). Analysis was performed with a FACS-Calibur (Becton Dickinson, Franklin Lakes, New Jersey, USA).

Statistics

Determination of significance between samples were made using the Student´s t test (GrapPad Prism; GraphPad Software). For all statistical tests, the 0,05 level of the confidence interval was accepted as statistically significant.

Figure legends

Figure 1 :

 1 Figure 1: Deletion of Fas in epidermal keratinocytes

Figure 2 :

 2 Figure 2: DNFB induced contact dermatitis in the absence of epidermal Fas expression a) 16 Fas E-KO mice (9 females and 7 males) and 14 control mice (6 females and 8 males) were

Figure 3 :

 3 Figure 3: Contact hypersensitivity induced keratinocyte apoptosis in Fas-negative

Figure 4 :

 4 Figure 4: Apoptosis of epidermal keratinocytes upon UVB-irradiation in Fas-negative

Figure 5 :Fas

 5 Figure 5: UVB-induced apoptosis of Fas-negative keratinocytes in vitro

Acknowledgements

This work was supported by a grant from the Medical Faculty, University of Cologne for A. H. (Koeln-Fortune program).

List of abbreviations