Coupling of oral human or porcine insulin to the B subunit of cholera toxin (CTB) overcomes critical antigenic differences for prevention of type I diabetes.

Our earlier investigations have demonstrated a critical difference in the efficacy of orally administered porcine compared to human or mouse insulin (no effect) in preventing type I diabetes in two distinct experimental models. Based on these findings one has to assume that certain insulins might not be suitable for the induction of oral 'tolerance'/bystander suppression, which might be one cause for recent failures in human oral antigen trials. Here we demonstrate that coupling to the non-toxic subunit of cholera toxin (CTB) can abolish these differences in efficacy between human and porcine insulin. As expected, an added benefit was the much smaller oral antigen dose required to induce CD4+ insulin-B specific regulatory cells that bystander-suppress autoaggressive responses. Mechanistically we found that uptake or transport of insulin-CTB conjugates in the gut occurs at least partially via binding to GM-1, which would explain the enhanced clinical efficacy. Both B chains bound well to major histocompatibility complex (MHC) class II, indicating comparable immunological potential once uptake and processing has occurred. Thus, our findings delineate a pathway to overcome issues in oral antigen choice for prevention of type I diabetes.

The cholera toxin B subunit directly costimulates antigen-primed CD4+ T cells ex vivo.

The nontoxic B subunit of cholera toxin (CTB) has been used as an adjuvant in experimental systems of mucosal vaccination. However, the mechanisms behind its adjuvant effects remain unclear. Here, we have used an ex vivo system to elucidate these mechanisms in antigen-specific T cells. Using splenocytes from keyhole limpet haemocyanin (KLH)-immunized mice, initial experiments showed that recombinant CTB (rCTB) did not affect the KLH-specific proliferation of splenocytes isolated from mice immunized 2 weeks earlier. However, rCTB strongly enhanced the KLH-specific proliferation of splenocytes from mice immunized with KLH 4 weeks prior. This adjuvant effect was dose-dependent, with maximum at 30-300 ng/ml rCTB. At higher doses of CTB this effect declined because of the induction of apoptosis. Using antibody depletion and coculture systems, we show that rCTB directly costimulates KLH-specific CD4+ and CD8+ T-cell proliferation but not B cells. Enzyme-linked immunospot (ELISPOT) assays revealed that rCTB also enhanced the KLH-specific CD4+ T-cell-mediated production of interleukin-2 (IL-2), IL-4 and interferon-gamma(IFN-gamma) by four to fivefold. Characterizing the adjuvant effect of rCTB in vivo confirmed the results above, i.e. rCTB mediated a twofold increase in the ex vivo T-cell response when used as a classical adjuvant in a secondary, but not in a primary KLH-immunization regimen. Together these data show that rCTB can act as a strong adjuvant, by directly costimulating antigen-primed CD4+ and CD8+ T cell in a dose-dependent manner. This new insight might be valuable in the future rational design of bacterial toxin-based vaccines.

The cholera toxin B subunit is a mucosal adjuvant for oral tolerance induction in type 1 diabetes.

When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model. In addition, the administration of this optimal insulin-CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.

No evidence for altered cellular immune functions in personnel deployed in the Persian Gulf during and after the Gulf War--The Danish Gulf War study.

Veterans who have participated in the Gulf War suffer from a number of symptoms, collectively referred to as the Gulf War Syndrome. It has been hypothesized that a change in the systemic cytokine balance or other changes in immunological parameters could be responsible for some of the symptoms. We analyzed the peripheral blood natural killer (NK) cell activity of 686 Gulf War personnel who had been present in the Persian Gulf area during and immediately after the Gulf War as well as 231 gender and age-matched controls. The test material included individual samples of frozen peripheral blood mononuclear cells kept at -139 degrees C for a period of 50 to 380 days prior to NK cell analysis of freshly thawed cells. Significant differences in NK-cell activity were not observed by direct comparison of the levels of natural cytotoxic activity in the two groups. However, NK-cell cytotoxicity as such decreased due to cryopreservation. Surprisingly, the NK cells obtained from control donors were significantly (p<0.0001) more sensitive to freezing conditions than cells from the Gulf War personnel, leaving the marginal comparison between the two groups untrustworthy, in particular because of the marked difference between the -139 degrees C storage times used for the two groups. Freshly thawed samples of peripheral blood T lymphocytes (CD2+ cells) from 109 randomly selected Gulf War personnel and 68 gender- and age-matched controls were stimulated for 3 days with phytohemagglutinin followed by 4 h activation by phorbol ester and ionomycin, and were stained for intracellular content of interleukin-2, -5, -10 and interferon-gamma. As with natural cytotoxicity, the length of cell storage at -139 degrees C influenced the production of cytokines. No significant differences in the cytokine production between the two groups were observed when the influence of the storage period was taken into consideration. Together, these data suggest that no overall long-term effects on NK-cell function and T-cell cytokine production are present in the Danish Gulf War personnel. Moreover, cryopreservation is a major potential source of bias when studying the physiology of thawed NK and T cells.

Stress-induced ClpP serine protease of Listeria monocytogenes is essential for induction of listeriolysin O-dependent protective immunity.

The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogen Listeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, a clpP deletion mutant of L. monocytogenes was used to study the generation of anti-Listeria protective immunity. We found that ClpP is required for the intracellular growth of L. monocytogenes in resident macrophages in vivo. Mice infected with doses as high as 10(6) clpP mutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disrupted hly gene, this lack of protection was overcome by inoculation of very high infecting doses of clpP mutant bacteria (5 x 10(8)), thus producing sufficient amounts of LLO to stimulate anti-Listeria immunity. The role of ClpP was confirmed by showing that anti-Listeria immunity was restored in mice infected with a clpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response against L. monocytogenes.

The majority of lamina propria CD4(+) T-cells from scid mice with colitis undergo Fas-mediated apoptosis in vivo.

We have previously shown that adoptively transferred CD4(+) T-cells mediate an chronic colitis in severe combined immune deficient (scid) mice. Colitis is accompanied by activation and apoptosis of Fas ligand and TNF-alpha expressing CD4(+) T-cells in the diseased colonic lamina propria (Eur. J. Immunol. 28:3655 (1998)). Here we investigate the apoptosis-inducing mechanism in these lamina propria infiltrating CD4(+) T-cells. We observe that freshly isolated lamina propria CD4(+) T-cells can kill Fas transfected P815 mastocytoma cells in a TCR/CD3 redirected chromium-release assay, but do not express TNF-alpha mediated cytotoxicity. Pre-incubation of the isolated lamina propria CD4(+) T-cells with an anti-FasL antiserum partially blocked killing of the Fas transfected target cells, indicating a role for the Fas-FasL system in the killing process. Treatment of scid mice with colitis with anti-FasL antiserum for 12 h blocked the apoptotic process in lamina propria CD4(+) T-cells by more than 65% compared to mice treated with control antiserum. Together, these results point towards the Fas-FasL and not the TNF-alpha-TNF-alpha receptor system as the primary apoptosis-inducing mechanism of lamina propria CD4(+) T-cells in this model of murine chronic colitis, and suggest an important role for the Fas-FasL system in the maintenance of homeostasis of locally proliferating T-cells.

Functional dichotomy in natural killer cell signaling: Vav1-dependent and -independent mechanisms.

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

Transforming growth factor-beta messenger RNA and protein in murine colitis.

Using a CD4+ T-cell-transplanted SCID mouse model of colitis, we have analyzed TGF-beta transcription and translation in advanced disease. By in situ hybridization, the epithelium of both control and inflamed tissues transcribed TGF-beta1 and TGF-beta3 mRNAs, but both were expressed significantly farther along the crypt axis in disease. Control lamina propria cells transcribed little TGF-beta1 or TGF-beta3 mRNA, but in inflamed tissues many cells expressed mRNA for both isoforms. No TGF-beta2 message was detected in either control or inflamed tissues. Immunohistochemistry for latent and active TGF-beta1 showed that all cells produced perinuclear latent TGF-beta1. The epithelial cell basal latent protein resulted in only low levels of subepithelial active protein, which co-localized with collagen IV and laminin in diseased and control tissue. Infiltrating cells expressed very low levels of active TGF-beta. By ELISA, very low levels (0-69 pg/mg) of soluble total or active TGF-beta were detected in hypotonic tissue lysates. TGF-beta1 and TGF-beta3 are produced by SCID mouse colon and transcription is increased in the colitis caused by transplantation of CD4+ T-cells, but this does not result in high levels of soluble active protein. Low levels of active TGF-beta may be a factor contributing to unresolved inflammation.

Conventional alpha beta T cells are sufficient for innate and adaptive immunity against enteric Listeria monocytogenes.

We have begun to dissect the cellular requirements for generation of immunity against enteric infection by Listeria monocytogenes using a novel T(-) B(-) NK(-) mouse strain (mice double deficient for the common cytokine receptor gamma-chain (gamma(c)) and the recombinase-activating gene-2 (RAG2/gamma(c) mice). Initial experiments showed that C57BL/6 mice and alymphoid RAG2/gamma(c) mice had similar kinetics of bacterial accumulation in the spleen, liver, and brain early after intragastric L. monocytogenes infection (up to day 3), calling into question the physiologic role of gut-associated lymphoid cells during the passage of this enterobacterium into the host. However, in contrast to C57BL/6 mice, RAG2/gamma(c) mice rapidly succumbed to disseminated infection by day 7. Polyclonal lymph node CD4(+) and CD8(+) alphabeta T cells were able to confer RAG2/gamma(c) mice with long-lasting protection against enteric L. monocytogenes infection in the absence of gammadelta T, NK, and NK-T cells. Moreover, these alphabeta T-reconstituted RAG2/gamma(c) mice produced IFN-gamma at levels comparable to C57BL/6 mice in response to L. monocytogenes both in vitro and in vivo. Protection was IFN-gamma dependent, as RAG2/gamma(c) mice reconstituted with IFN-gamma-deficient alphabeta T cells were unable to control enteric L. monocytogenes infection. Furthermore, alphabeta T cell-reconstituted RAG2/gamma(c) mice were able to mount memory responses when challenged with lethal doses of L. monocytogenes. These data suggest that NK, NK-T, gammadelta T, and B cells are functionally redundant in the immunity against oral L. monocytogenes infection, and that in their absence alphabeta T cells are able to mediate the early IFN-gamma production required for both innate and adaptive immunity.

The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis.

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

Cells and cytokines in the pathogenesis of inflammatory bowel disease: new insights from mouse T cell transfer models.

Recently, a number of experimental models of human inflammatory bowel disease (IBD) of immunological basis have been developed. These have proven useful tools in the study of IBD, allowing a more detailed dissection of the pathogenesis of the disease. Studies from these models have revealed new, important knowledge about environmental factors, cell subset, cytokines and effector molecules in the pathogenesis of IBD. This review focuses on recent advances in the understanding of the development of IBD obtained from adoptive CD4+ T cell transfer models of the disease.

Deficiency of the intestinal growth factor, glucagon-like peptide 2, in the colon of SCID mice with inflammatory bowel disease induced by transplantation of CD4+ T cells.

BACKGROUND: Glucagon-like peptide 2 (GLP-2) is produced in endocrine L-cells of the intestinal mucosa. Recently, GLP-2 was found to stimulate intestinal mucosal growth. Our objective was to study the content of GLP-2 in the large intestine in a murine model of T-cell-induced inflammatory bowel disease. METHODS: Inflammation was induced by adoptive transfer of CD4+ blast T cells from BALB/c mice to SCID mice. The amount of GLP-2 (1-33) was measured with a specific, NH2-terminally directed radioimmunoassay in tissue extracts from the large intestine of transplanted mice developing colitis and from BALB/c and SCID control mice. RESULTS: In the middle and descending colon segments showing the most severe signs of inflammatory lesions in the CD4+ T-cell-transplanted mice, the amount of GLP-2 was significantly lower than in similar colon segments in both untransplanted SCID mice and normal BALB/c mice (P = 0.0013 and 0.0033). In the descending colon the amount of GLP-2 was 6.7 +/- 1.0 pmol/g protein in the CD4+ transplanted mice compared with 68.4 +/- 20.3 and 42.7 +/- 4.3 in the two groups of control mice. Similar findings were made with regard to the contents of the two other proglucagon-derived intestinal peptides, glicentin and GLP-1. CONCLUSION: The amount of GLP-2 is markedly reduced in the colon of mice with a T-cell-induced inflammatory bowel disease histopathologically resembling both Crohn disease and ulcerative colitis. This observation may provide a pathophysiologic rationale for administration of GLP-2 as a trophic factor in inflammatory bowel disease.

The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes.

We identified the stress-induced ClpP of Listeria monocytogenes and demonstrated its crucial role in intracellular survival of this pathogen. ClpP is a 21.6 kDa protein belonging to a family of proteases highly conserved in prokaryotes and eukaryotes. A clpP-deleted mutant enabled us to demonstrate that ClpP is involved in proteolysis and is required for growth under stress conditions. Intramacrophage survival of this mutant was strongly restricted, thus resulting in loss of virulence for the mouse. The activity of listeriolysin O, a major virulence factor implicated in bacterial escape from phagosomes of macrophages, was much reduced in the clpP mutant under stress conditions. Direct evidence for the role of ClpP in the intracellular parasitism was obtained by showing that virulence and haemolytic activity were fully restored by complementation of the mutant. These results suggest that ClpP is involved in the rapid adaptive response of intracellular pathogens during the infectious process.

In vitro activated CD4+ T cells from interferon-gamma (IFN-gamma)-deficient mice induce intestinal inflammation in immunodeficient hosts.

To investigate the role of IFN-gamma in the immunopathogenesis of inflammatory bowel disease (IBD), severe combined immunodeficient (SCID) mice were transplanted with in vitro activated CD4+ T cells from either wild-type (WT) or IFN-gamma-deficient (IFN-gammaKO) BALB/c mice. In vitro, the two types of T cells displayed comparable proliferation rates and production of tumour necrosis factor-alpha (TNF-alpha), IL-2, IL-4 and IL-10 after concanavalin A (Con A) stimulation. When transplanted into SCID mice, WT CD4+ blasts induced a lethal IBD, whereas IFN-gammaKO blasts induced a less severe intestinal inflammation with moderate weight loss. Intracellular cytokine staining of lamina propria lymphocytes (LPL) revealed comparable fractions of CD4+ T cells positive for TNF-alpha, IL-2 and IL-10 in the two groups of transplanted SCID mice, whereas a two-to-three-fold increase in the fraction of IL-4-positive cells was found in IFN-gammaKO-transplanted SCID mice. Flow cytometric analyses showed strong up-regulation of MHC class II expression of colonic epithelial cells of WT-CD4+ T cell-transplanted compared with IFN-gammaKO-transplanted SCID mice. A significantly higher fraction of CD4+ LPL were found to enter the cell cycle, i.e. to incorporate bromo-deoxy-uridine, and to undergo apoptosis in vivo in WT-transplanted compared with IFN-gammaKO-transplanted SCID mice. These data point towards an important role for IFN-gamma in the development of IBD in SCID mice. The inflammation might be initiated and subsequently enhanced by the ability of IFN-gamma to induce de novo MHC class II expression in the colonic epithelium, a change which could lead to increased antigen processing and production of local proinflammatory cytokines, CD4+ T cell turnover and thereby to exaggeration of disease.

Signal transduction by the major histocompatibility complex class I molecule.

Ligation of cell surface major histocompatibility class I (MHC-I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC-I-mediated signaling can lead to both increased and decreased activity of the MHC-I-expressing cell depending on the fine specificity of the anti-MHC-I antibodies, the context of CD8 ligation, the nature and cell cycle state of the MHC-I-expressing cell and the presence or absence of additional cellular or humoral stimulation. This paper reviews the biochemical, physiological and cellular events immediately after and at later intervals following MHC-I ligation. It is hypothesized that MHC-I expression, both ontogenically and in evolution, is driven by a cell-mediated selection pressure advantageous to the MHC-I-expressing cell. Accordingly, in addition to their role in T-cell selection and functioning, MHC-I molecules might be of importance for the maintenance of cellular homeostasis not only within the immune system, but also in the interplay between the immune system and other organ systems.

MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases.

In addition to providing the framework for peptide presentation, major histocompatibility complex class I (MHC-I) molecules can act as signal transducing molecules in lymphoid cells. Here we show that the mobilization of intracellular calcium, which follows crosslinking of MHC-I molecules on human B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine/threonine kinases in MHC-I-mediated apoptosis in human B-cells and suggest the presence of several MHC-I signaling pathways leading to diverse effects in these cells.

Tumour necrosis factor-alpha (TNF-alpha) transcription and translation in the CD4+ T cell-transplanted scid mouse model of colitis.

The adoptive transfer of activated CD4+ alpha/beta T cell blasts from the spleens of immunocompetent C.B-17+/+ or BALB/cdm2 mice into C.B-17scid/scid (scid) mice induces a colitis in the scid recipient within 8 weeks, which progresses to severe disease within 16 weeks. T cells isolated from recipient colon show a Th1 cytokine phenotype. We have examined the relationship between the phenotype of the cellular infiltrate and the transcription and translation of the proinflammatory cytokine TNF-alpha. The techniques of double indirect immunohistology and in situ hybridization using digoxigenin-labelled riboprobes were used. The prominent myeloid cell infiltrate in diseased tissues comprised F4/80+, Mac-l+ macrophages, neutrophils, dendritic cells and activated macrophages. TNF-alpha transcription and translation were associated with activated macrophages in the lamina propria. Activated macrophages transcribing and translating TNF-alpha were clustered in areas of tissue destruction. Crypt epithelium of inflamed tissues transcribed TNF-alpha at a very early stage of the disease process, but translation of TNF-alpha protein could only be found in advanced epithelial dysplasia. This indicates differential post-transcriptional control of TNF-alpha in activated macrophages and the epithelium.

Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype.

We studied the induction, severity and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6-12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12-16 wk posttransfer. SCID mice transplanted with STAT-4-/- CD4+ T cells showed a late-onset IBD manifest > 20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-gamma and TNF-alpha. In SCID mice transplanted with IL-12-unresponsive STAT-4-/- CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-gamma but abundant levels of TNF-alpha. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency.

The CC-chemokine receptor 5 (CCR5) is a marker of, but not essential for the development of human Th1 cells.

The CC-chemokine receptor 5 (CCR5) has recently been described as a surface marker of human T cells producing type 1 (Th1) cytokines. Here we confirm that CCR5 is expressed on human Th1 but not on Th2 T-cell clones. Using intracellular cytokine staining, we show that alloantigen specific CD4+ T-cell lines derived from a CCR5-deficient individual (delta32 allele homozygote) contain high numbers of both interferon gamma (IFN-gamma) and interleukin (IL)-2 producing cells, low numbers of IL-10 producing cells and no IL4 or IL-5 producing cells when stimulated with phorbol ester and ionomycin in vitro. These results were similar to those obtained from alloantigen specific CD4+ T-cell lines derived from CCR5 expressing individuals. An enzyme-linked immunoabsorbent assay (ELISA) confirmed that the Th1 cytokine-positive cells from the CCR5-deficient individual were able to produce equal amounts of cytokines when compared to T-cell lines from CCR5-expressing individuals, These results demonstrate that CCR5-negative T cells display the same capacity of Th1 T-cell differentiation as T cells derived from CCR5-expressing individuals. Thus, CCR5 expression is not essential for differentiation of human Th1 T cells.

Proliferation and apoptosis of lamina propria CD4+ T cells from scid mice with inflammatory bowel disease.

Scid mice transplanted with low numbers of syngeneic CD4+ T cells, develop a chronic and lethal inflammatory bowel disease (IBD) within 4-6 months. We have used in vivo 5-bromo2-deoxy-uridine (BrdU) labeling to assess the proliferation of lamina propria-derived CD4+ T cells in diseased scid mice. The hourly rate of renewal of colonic lamina propria CD4+ T cells in diseased mice was 7% compared with 1.5% in normal BALB/c control mice. Transplantation of scid mice with in vitro activated CD4+ T cells accelerated the disease onset and development in a cell dose-dependent fashion when compared with non-activated CD4+ T cells. In pulse-chase experiments it was shown that BrdU-labeled cells disappeared rapidly from the lamina propria of diseased mice. DNA analysis revealed that this was due to the presence of nearly four times as many apoptotic CD4+ T cells in diseased than in control mice. Further analyses showed that the apoptotic lamina propria CD4+ T cells were derived from cells having entered the cell cycle within the previous 8 h. These data clearly demonstrate that vigorous CD4+ T cell proliferation and death are involved throughout the course of IBD.