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Distinct Requirements for C-C Chemokine and IL-2 Production by Naive, Previously Activated, and Anergic T Cells

Cara G. Lerner^*, Maureen R. Horton, Ronald H. Schwartz^* and Jonathan D. Powell^1,*

^* Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and Pulmonary Division, Johns Hopkins University School of Medicine, Baltimore, MD 21205

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ag presented by activated APCs promote immunogenic responses whereas Ag presented by resting APCs leads to tolerance. In such a model, the regulation of cytokine release by the presence or absence of costimulation might potentially play a critical role in dictating the ultimate outcome of Ag recognition. C-C chemokines are a structurally defined family of chemoattractants that have diverse effects on inflammation. We were interested in determining the activation requirements for chemokine production by CD4⁺ T cells. Our data demonstrate for T cell clones and previously activated T cells from TCR-transgenic mice that stimulation with anti-TCR alone results in the production of copious amounts of macrophage-inflammatory protein-1 (MIP-1) and other C-C chemokines, and that addition of anti-CD28 gives very little augmentation. Furthermore, MIP-1 production is nearly equivalent from both anergic and nonanergic cells. For naive T cells, anti-CD3 stimulation alone led to as much MIP-1 production as Ag + APC stimulation. The addition of costimulation gave a 3–10-fold enhancement, but this was 70-fold less than the effect of costimulation on IL-2 production. Thus, although C-C chemokines play a broad role in influencing inflammation, their production by signal 1 alone makes them unlikely to play a critical role in the decision between a tolerogenic and an immunogenic response. Furthermore, the production of MIP-1 by anergic T cells, as well as following signal 1 alone, raises the possibility that in vivo this chemokine serves to recruit activated T cells to become tolerant.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Athough a number of molecules act to enhance T cell activation, for example, LFA-1/ICAM-1, and CD-2/LFA-3 (1, 2), true costimulation may be best characterized as a distinct separate signal that is necessary, even in the presence of maximal TCR signaling, to produce a proliferative response. Our laboratory has been examining the consequences of TCR engagement in the presence and absence of costimulation. Using pigeon cytochrome c (PCC)²-specific CD4⁺ Th1 T cell clones, we have defined a consistent hierarchy of costimulatory requirements for cytokine production: IL-2 > IL-3 > IFN-. That is, IFN- is produced in response to TCR engagement alone and increases only about 2-fold upon the addition of costimulation. To a lesser degree, IL-3 is also produced upon TCR stimulation and there is an 3-fold increase with the addition of costimulation. IL-2, however, demonstrates a near absolute requirement for both TCR engagement and costimulation (signals 1 + 2) (3). Indeed, in the absence of costimulation, these cells enter a state of hyporesponsiveness termed anergy. The hallmark of T cell clonal anergy is a decrease in proliferation as a consequence of a marked decrease in IL-2 secretion upon full restimulation (signals 1 + 2) (4, 5). On the other hand, anergic cells still produce IFN- and, to a lesser extent, IL-3 (6, 7). In fact, the hierarchy of costimulation dependence is mirrored by the hierarchy of cytokine inhibition in anergy. For example, IL-2 has the greatest dependence on costimulation for its production and is also the most inhibited in anergy, whereas IFN- production is the least dependent on costimulation and the least affected by the anergic state.

The C-C chemokines are a structurally defined family of small proteins in the chemokine superfamily. Characterized by macrophage-inflammatory protein-1 (MIP-1), MIP-1ß, RANTES, and monocyte chemoattractant protein-1 (MCP-1), these proteins were principally defined as chemoattractants for monocytes, eosinophils, basophils, and lymphocytes (8, 9, 10, 11). It is becoming increasingly clear, however, that chemokines not only act as chemoattractants in the inflammatory response, but also play a role in specifically enhancing T cell responses (12, 13, 14, 15). Although much is known about the requirements for chemokine production in macrophages and monocytes, the precise activation requirements for secretion by T cells are less clear. Using murine T cells, Herold et al. (16) recently reported that murine C-C chemokine production was dependent on CD28 signaling, suggesting a necessity for costimulation. In contrast, using human PBL, others were able to demonstrate MIP-1 production by stimulation with anti-CD3 alone (17), whereas another group showed that MIP-1 and MIP-1ß production was dependent on the addition of PMA to the anti-CD3 (18).

In light of the emerging importance of chemokines in mediating an immune response, we were interested in determining the signaling requirements for C-C chemokine production, their production by anergic cells, and their place in our hierarchy of cytokine production and inhibition. Using a murine Th1 CD4⁺ T cell clone as well as in vitro activated TCR-transgenic T cells (subsequently termed previously activated T cells), we found that TCR engagement alone results in the secretion of copious amounts of C-C chemokines even when the cells were in an anergic state. For naive T cells, signal 1 alone also proved to be sufficient to produce MIP-1; however, the addition of anti-CD28 did lead to an enhancement of this response. In contrast, IL-2 production from the same cultures demonstrated a near absolute requirement for costimulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines and culture conditions

A.E7 is a CD4⁺ Th1 clone specific for the PCC peptide 81-104 and was grown and maintained as described previously (19). In general, cells were stimulated for 48 h with whole PCC in the presence of irradiated (3000 rads) B10.A splenocytes as a source of APC. The A.E7 cells were then expanded 20-fold in 10 U/ml of rIL-2. After a minimum of 12 days in culture to allow the cells to rest down, live cells were isolated on a Ficoll gradient and utilized. Cells were maintained in medium consisting of equal volumes of RPMI 1640 and Eagle’s modified Hanks’ amino acids (Biofluids, Rockville, MD) supplemented with 10% FCS, 5 x 10^-5 M 2-ME, 4 mM glutamine, and antibiotics at 37°C in a 5% CO₂ humidified incubator.

Fresh cell purification and stimulation

CD4⁺ naive splenocytes or lymph node cells were isolated from Rag-2^-/-, B10.A TCR 5CC7-transgenic mice (National Institute of Allergy and Infectious Diseases, Bethesda, MD/Taconic Farms, Germantown, NY), expressing a TCR specific for PCC peptide 81-104 presented in the context of I-E^k, by negative selection through MidiMacs column (Miltenyi Biotec, Auburn, CA) separation. In brief, spleens were harvested, RBC were lysed with ACK lysing buffer (Biofluids), and cells were incubated for 30 min on ice with monoclonal rat anti-mouse MHC class II (Ia) (clone: M5/114.15.2), monoclonal rat anti-mouse CD8a (Ly-2) (clone 53-6.7), and monoclonal hamster anti-mouse CD11c (clone N418) Abs conjugated to super paramagnetic microbeads (Miltenyi Biotec). Cells were then washed with 1% FCS in PBS and passed through an LS⁺ MidiMacs Separation column (Miltenyi Biotec) placed in the magnetic field of a magnetic cell separation system separator (Mitenyi Biotech). Positively selected cells were discarded, and negatively selected cells were washed with 1% FCS in PBS. An aliquot was stained with PE anti-mouse CD44 (clone IM7), APC anti-mouse CD4 (clone RM4-5), and FITC anti-mouse CD8 (clone 53-6.7; PharMingen, San Diego, CA) to determine the purity of the population. To generate CD4⁺CD44^high previously activated cells, an aliquot of the CD44^low cells was stimulated, like A.E7 cells, with irradiated B10.A splenocytes plus PCC for 48 h. The cells were then expanded 10-fold in 5 U/ml rIL-2 and were utilized 5–7 days later after isolation on a Ficoll gradient.

Cell stimulation for cytokine production

Conditions were determined for optimal IL-2 production by the clones or fresh cells. A.E7 cells were stimulated in 24-well plates (5 x 10⁵ cells/well) in a total volume of 0.5 ml. Some wells were precoated with 10 µg/ml of anti-TCR-ß Ab H57-597 (20). Costimulation was provided by adding ascitic fluid containing the anti-CD28 mAb 37.51 (21) at a final dilution of 1:5000. Other wells were treated with PMA alone (Sigma, St. Louis, MO) at a final concentration of 0.25ng/ml, ionomycin alone (Sigma) at a final concentration of 4 µM, or the two together. In addition, in some cases the drugs cyclosporin A (CSA) (Calbiochem, Cambridge, MA) at a final concentration of 1 µM, proteasome inhibitor 1 (Calbiochem) at a final concentration of 2 µM, and/or PD90859 (New England Biolabs, Beverly, MA) at a final concentration of 50 µM were added to these cultures. Supernatant fluids were harvested after 16 h and frozen at -20°C until used. IL-2 production was assessed by measuring the proliferation of the CTL-L cell line (American Type Culture Collection, Manassas, VA) (22) as described previously (23). IL-2 determination was calculated from 8 twofold serial dilutions. In other experiments, to facilitate processing of samples, IL-2 was determined by ELISA (R&D Systems, Minneapolis, MN) using multiple serial dilutions. In our laboratory, 1 U/ml of IL-2 as measured in the CTL-L assay corresponds to 1 pg/ml of IL-2 in the ELISA. MIP-1 and RANTES production were assayed by ELISA (R&D Systems) in a similar fashion to IL-2. All figures are representative of more than three experiments. The means and errors of the mean, as well as the statistical significance of the differences between the two means, usually derived from log transformation of the data and assessed by Student’s t test, are found in the text of the Results.

Fresh naive and previously activated cells were stimulated similar to the A.E7 cells; however, these cells were stimulated in 24-well plates with 1 x 10⁶ cells/well in 0.5 ml of medium, and supernatants were harvested after 48 h. In some cases, these cells were stimulated in wells precoated with anti-CD3 (PharMingen clone 2C11) at a concentration of 10 µg/ml. In addition, stimulation was also conducted by using irradiated B10.A splenocytes at a concentration of 2.5 x 10⁶/well with 1 µM of the PCC peptide 81-104.

Northern blot analysis

A.E7 cells were mock stimulated, stimulated with 10 µg/ml precoated anti-TCR, or stimulated with precoated anti-TCR and soluble anti-CD28 at a final concentration of 1:5000 in a 6-well plate for 8 h. The cells were then harvested and lysed in guanine isothiocyanate and Northern blot analysis was performed as described previously (24).

Anergy induction

Anergy was induced as described previously (3). Briefly, 10⁸ A.E7 cells were stimulated overnight in a 162-cm² tissue culture flask precoated with 10 µg/ml anti-TCRß. To induce anergy in the 5CC7 primary effector cells, 5 x 10⁷ cells were stimulated overnight in a T75 tissue culture flask precoated with 10 µg/ml anti-TCR. Cells were then harvested using a sterile cell scraper, washed, and rested in fresh medium. Anergic T cells demonstrate decreased proliferation to their cognate Ag upon restimulation as a result of their marked decrease in IL-2 production (3, 25). We have found that the decreased production of IL-2 is a more quantitative measure of anergy induction and thus throughout this paper anergy is assessed in terms of decreased IL-2 production.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MIP-1 is produced by anergic T cells

Our laboratory has observed a consistent hierarchy of cytokine production by Th1 CD4⁺ T cell clones, whereby the more dependent a cytokine is on costimulation for production, the greater its inhibition in anergy. Previously, it was shown that MIP-1 production by T cells required costimulation in the form of CD28 signaling (16). According to the hierarchy then, we would predict that anergic cells would not produce MIP-1. To test this hypothesis, anergic A.E7 T cells were stimulated for 16 h in fresh medium with anti-TCR + anti-CD28. Supernatant fluids were analyzed by ELISA for MIP-1 or IL-2 production. As seen in Table I, there was a profound inhibition of IL-2 production (>100-fold) in the anergic A.E7 cells, indicating that they were indeed in an anergic state. Surprisingly, however, the cells produced similar amounts of MIP-1 as compared with their nonanergic counterparts. In six experiments, the anergic cells showed an average of only a 1.34-fold decrease in MIP-1 production as compared with nonanergic cells (paired Student’s t test, p = 0.11). Also, surprisingly, the unstimulated anergic clones continued to secrete a small yet consistent amount of MIP-1 after anergy induction. This expression, which appears to be due to newly transcribed protein, was not cumulative, but rather was freshly produced during the 16-h assay period. Small amounts of secretion were observed even up to 9 days after anergy induction. This continued expression of MIP-1 after anergy induction could be inhibited by the addition of fresh IL-2. In one experiment, anergic A.E7 cells produced 1333 pg/ml MIP-1 during a 16-h period on day 4 after anergy induction. This was decreased to 10.7 pg/ml by the addition of 100 U/ml IL-2. On the other hand, IL-2 does not inhibit MIP-1 production by cells that are actively being stimulated. For example, A.E7 cells produced 168,500 pg/ml of MIP-1 in response to stimulation and 280,000 pg/ml in response to stimulation in the presence of 100 U/ml IL-2. These findings are consistent with our observations that stimulation with signals 1 + 2 leads to the production of copious amounts of both IL-2 and MIP-1 (see below); the IL-2 produced under these conditions does not inhibit MIP-1 production.

View this table: [in this window] [in a new window]   Table I. Anergic T cell clones produce MIP-11

Since MIP-1 production was not affected by the anergic state, we decided to examine the costimulation dependence of this chemokine in our Th1 clone. As demonstrated by Northern blot analysis in Fig. 1, stimulation by anti-TCR for 8 h led to a dramatic increase in MIP-1, MIP-1ß, and, to a lesser extent, RANTES mRNA. MCP-1 mRNA was undetectable (note different exposure times). In fact, preliminary data using microarray technology (26) (data not shown) has demonstrated that MIP-1 is one of the most abundant transcripts up-regulated in these T cell clones upon TCR engagement alone. The addition of anti-CD28 appeared to have minimal effect on all four chemokines, suggesting that A.E7 TCR engagement is sufficient for the maximum chemokine expression obtainable with this clone.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Up-regulation of C-C chemokine mRNA by CD4+ Th1 clones does not require costimulation. A, E7 cells were mock stimulated or stimulated with anti-TCR or anti-TCR and anti-CD28 for 8 h. The cells were then harvested and RNA was examined by Northern blot analysis.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. MIP-1 production by CD4+ T cell clones does not require costimulation. A, E7 cells were mock stimulated, stimulated with anti-TCR or anti-TCR plus anti-CD28 for 16 h, and supernatant fluids were assayed by CTL-L for IL-2 production (A) or ELISA for MIP-1 production (B) and RANTES (C). All three cytokines were assayed from the same supernatant fluid.

MIP-1 production is dependent on signaling through the Ca²⁺/calcinuerin, NF-B, and extracellular signal-related kinase (ERK) kinase pathways

Because TCR stimulation alone resulted in the production of C-C chemokines, we were interested in determining which TCR-mediated signaling pathways were responsible. TCR engagement leads to activation of the Ca²⁺/calcineurin pathway, the ERK kinase pathway, and NF-B family members (reviewed in Ref. 27). We measured chemokine production in the presence of pharmacologic agents that selectively stimulate or inhibit these specific pathways. One representative experiment is shown in Table II. PMA alone, which activates protein kinase C and leads to the activation of RAS and NF-B proteins (27) essentially stimulated no MIP-1 (Table II) or RANTES (data not shown). On the other hand, ionomycin, which stimulates the Ca²⁺/calcineurin pathway (27), resulted in a modest yet significant amount of MIP-1 production. This effect was consistently observed (3500 pg/ml ± 1400 in seven experiments) in spite of the fact that neither IL-2 nor RANTES (data not shown) was induced by ionomycin alone. In contrast, the addition of PMA and ionomycin together had a synergistic effect, virtually recapitulating the levels of MIP-1 secreted upon TCR engagement. These results were obtained with the optimal drug concentrations determined by dose-response curves for both reagents.

View this table: [in this window] [in a new window]   Table II. Signal 1-induced MIP-1 production utilizes the Ca2+, ERK kinase, and NF-B pathways1

MIP-1 production in stimulated and anergized previously activated CD4⁺ T cells from TCR-transgenic mice mimics that of the T cell clones

To generalize our findings to freshly isolated cells, CD4⁺ T cells were prepared by negative selection from splenocytes of B10.A 5CC7 TCR-transgenic Rag-2^-/- mice specific for the PCC peptide 81-104 in the context of I-E^k. As represented in Fig. 3A, negatively separated naive cells were 95% CD4⁺, CD44^low, or CD44^int. The small percentage of CD44^high cells were not CD4⁺ (data not shown). An aliquot of these cells was assayed as the naive population, while a portion was used for generating CD44^high cells in vitro (termed previously activated cells). The latter were generated by stimulating the naive cells with peptide and APC for 48 h. The cells were then expanded in 5 U/ml of IL-2 and rested for 5–7 days before use. As seen in Fig. 3B, the activated population showed a shift in CD44 expression to mostly CD44^high, confirming that they had been activated by Ag. Both the naive and previously activated cells were mock stimulated and stimulated with anti-TCR or anti-TCR and anti-CD28. Supernatant fluids were then analyzed for cytokine production.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. Flow cytometry analysis of freshly isolated Rag-2-/- B10.A TCR 5CC7 spleen cells. A, CD4+ splenocytes were isolated by negatively selecting MHC class II+ and CD11c+ cells by magnetic bead separation. Cells used in the assay were 94.7% CD4+ and 92% CD44low. B, Previously activated cells were generated by in vitro stimulation of the naive CD4+ cells with peptide and APC. A complete shift of the population to CD44high can be seen, indicating that the cells had been activated.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. MIP-1 production by freshly isolated, CD4+, previously activated T cells does not require costimulation. Previously activated cells were generated as shown in Fig. 3. Some of the cells were induced into an anergic state by stimulation with plate-bound anti-TCR for 16 h, followed by 5 days of rest. The cells were then stimulated with anti-TCR or anti-TCR + anti-CD28 or left unstimulated (mock). The supernatant fluid was collected and assayed for IL-2 (A and C) or MIP-1 (B and D).

MIP-1 production by naive T cells

The lack of a significant effect of CD28 costimulation on MIP-1 production by previously activated TCR-transgenic T cells and T cell clones differs from the observations of Herold et al. (16). Their studies, however, were conducted using freshly isolated T cells and anti-CD3 or Ag as a stimulus. Therefore, to mimic their conditions, fresh naive T cells isolated from B10.A 5CC7 TCR-transgenic Rag-2^-/- mice were stimulated as shown in Table III. Stimulation with PMA and ionomycin induced large amounts of MIP-1 as well as IL-2, demonstrating the ability of the naive T cells to produce both molecules. Likewise, stimulation of these cells by APC plus peptide resulted in significant production of MIP-1 and IL-2. Interestingly, the anti-CD3 alone induced as much MIP-1 as APC plus peptide. In this experiment, the addition of anti-CD28 led to a 2.9-fold increase. This ability of costimulation to enhance MIP-1 production was consistently observed, as seen by the kinetics experiment depicted in Fig. 5A and a third experiment shown in Fig. 5B. Overall, for six experiments, the mean fold augmentation produced by the addition of anti-CD28 was 5-fold (Student’s t test on log transformed data, p = 0.003). Even so, by contrast, the effect of costimulation on IL-2 production was much greater. For the experiment shown in Table III, the effect was 200-fold and for the experiment depicted in Fig. 5B the effect was 700-fold. For both experiments, the costimulatory effect of anti-CD28 was about 70-fold greater for IL-2 production than for MIP-1 production. Overall, these data demonstrate that under optimal stimulation conditions (48 h) anti-CD3 alone is sufficient to elicit large amounts of MIP-1 from naive T cells. In contrast to what is seen for T cell clones and previously activated cells, the addition of anti-CD28 can augment this production; however, this effect is small when compared with the costimulatory effect seen for IL-2 production.

View this table: [in this window] [in a new window]   Table III. MIP-1 production by naive T cells1

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Costimulation enhances MIP-1 production but is required for IL-2 production. Naive CD4+ T cells from Rag-2-/- B10.A 5CC7 mice were stimulated with plate-bound anti-CD3 or anti-CD3+ anti-CD28. A, Supernatant fluid was harvested at the indicated time points and assayed for MIP-1. B, Cells were stimulated for 48 h, after which supernatant fluids were harvested and assayed for MIP-1 and IL-2 content by ELISA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In our in vitro system, plate-bound anti-CD3 alone was able to induce substantial quantities of MIP-1 even in naive cells. This is in contrast to the observations of Herold et al. (16) who reported that anti-CD3 alone was not sufficient to induce MIP-1 production. In their experiments, however, MIP-1 production was only measured at 16 h. Although this time point provides optimal stimulation for T cell clones, we have found that naive T cells produce only one-tenth the amount of MIP-1 at 16 h that they produce at 48 h, even under optimal stimulation conditions (Fig. 5A). In addition, even at the early time point in our system, cells stimulated with anti-CD3 alone produced as much MIP-1 as cells stimulated with anti-CD3 + anti-CD28 in their system, perhaps indicating that their stimulating conditions were not designed to induce maximal TCR stimulation. In this context, we would propose that in their experiments they were using a relatively naive population of T cells, and that under suboptimal stimulation conditions, the addition of anti-CD28 may have served to enhance the transduction of signal 1 as opposed to providing a distinct and necessary second signal. Likewise, when they added blocking Abs to CD28, they might have been decreasing ligand interactions involved in transducing signal 1. Such signaling could facilitate raft microdomain formation as has recently been described (37). We have also observed that the addition of anti-CD28 only enhances MIP-1 production in naive cells and not in previously activated T cells and T cell clones. Critically, the costimulatory enhancement of MIP-1 production by naive cells did not reflect an absolute requirement for costimulation. This is in contrast to IL-2 production which requires a distinct second signal, i.e., costimulation, as originally defined by Lafferty and Cunningham (38). Notably, the two-signal requirement for the production of IL-2 holds for naive cells, previously activated cells, and T cell clones.

That MIP-1, which does not require costimulation, is produced comparably by both anergic and nonanergic cells, fits with our previous observations on the similarities between the hierarchy of cytokine production with costimulation and in anergy. Anergy is characterized by a block in the MAP kinase pathway at the level of RAS (39, 40). It is presumed that this block leads to a decrease in the production and activation of AP-1 transcription factors, which in turn leads to a decrease in IL-2 production. In A.E7 T cell clones, however, TCR-induced MIP-1 production is PD90859 sensitive, suggesting that this transcription is dependent on the MAP kinase pathway. Thus, a paradox exists since MIP-1 production is minimally reduced in stimulated anergic T cells. One possible resolution is that the block in MEK1 activity in anergic cells is leaky such that the remaining activity of MEK1 is enough to drive transcription of MIP-1 but not IL-2. Another possibility is that PD90859 affects other unknown kinases that can lead to MIP-1 production but not IL-2 production. Finally, a third possibility, which we favor, is that the profound decrease in the production of IL-2 in anergic cells is not due solely to a block in the MAP kinase pathway, but the result of active repression at the level of the IL-2 promoter. Our laboratory has previously identified a site centered around the -180 region of the IL-2 promoter as a target for transcriptional repression (23). More recent studies showed that cAMP-responsive binding protein/cAMP-responsive modulator complexes bind to this site and promote the repression of transcription (25). Based on the observation that both IL-2 and MIP-1 are inhibited by PD90859, but only IL-2 is inhibited in anergy, we would argue that the mediators of transcriptional repression in anergy do not bind to the MIP-1 promoter.

The regulation of MIP-1 production is strikingly similar to the induction of anergy in T cells. Both T cell anergy and MIP-1 production are induced by TCR stimulation alone, as well as partially by stimulation with ionomycin alone. Both are inhibited by CSA. In addition, IL-2 prevents the induction of anergy and leads to its reversal, whereas IL-2 inhibits the low level of continuous production of MIP-1 seen by anergic cells. Finally, the long-lasting nature of T cell anergy suggests that the negative factors responsible for this state are constitutively expressed long after TCR stimulation is completed. Likewise, low levels of constitutive expression of MIP-1 are observed long after stimulation through the TCR. Although MIP-1 itself does not induce anergy (data not shown), delineating the pathways involved in the expression of MIP-1 may provide insight into the factors necessary for the maintenance of the anergic state.

A role for C-C chemokines is emerging in contributing to the pathogenesis of a number of autoimmune disorders. For example, murine experimental autoimmune encephalomyelitis is characterized by elevated levels of MIP-1, MIP-1ß, MCP-1, and RANTES in the CNS (41, 42, 43), whereas in autoimmune diabetes, secretion of C-C chemokines is thought to attract Th1 cells to pancreatic islets (44). Likewise, chemokine dysregulation has been found in patients with inflammatory bowel disease (45). Interestingly, inflammatory bowel disease is a prominent component of the autoimmunity seen in the IL-2 knockout mouse (46). In light of our observation that IL-2 shuts off the continuous expression of signal 1-induced MIP-1, perhaps a component of the strong inflammation in the gut is due to dysregulation of chemokine production in the absence of IL-2. Finally, in light of the fact that anergic T cells produce MIP-1, it may be that in the absence of costimulation, these T cells recruit other Ag-specific cells to become tolerized. In this case, to promote tolerance, a stimulated anergic T cell would attract Ag-activated T cells (which express C-C chemokine receptors) and/or APC to a particular site where they would be stimulated by signal 1 in the absence of costimulation. Such a mechanism might serve to promote linked suppression (47). It has recently been shown in a rat model that anergic T cells can suppress APC function (48). Thus, just as chemokine production by activated T cells might serve to attract cells to the sight of inflammation, chemokine secretion by anergic T cells may serve to suppress potential autoimmune disease.

	Acknowledgments

 
We thank Dr. B. J. Fowlkes and Dr. Joshua Farber for their critical review of this manuscript. In addition, we thank our colleagues in the Laboratory of Cellular and Molecular Immunology for their helpful suggestions and support.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Jonathan D. Powell, Laboratory of Cellular and Molecular Immunology, Building 4, Room 111, National Institutes of Health, Bethesda, MD 20892-0420.

² Abbreviations used in this paper: PCC, pigeon cytochrome c ; MIP-1, macrophage-inflammatory protein-1; MCP-1, monocyte chemoattractant protein-1; CSA, cyclosporin A; ERK, extracellular signal-related kinase; MAP, mitogen-activated protein; MEK, mitogen-activated ERK kinase.

Received for publication October 21, 1999. Accepted for publication February 2, 2000.

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