Neuroglial response after induction of experimental allergic encephalomyelitis in susceptible and resistant rat strains

Experimental allergic encephalomyelitis (EAE), the animal model for multiple sclerosis in humans, a T-cell mediated disease of the central nervous system is characterized by inflammatory infiltrates of myelin antigen(s)-specific T cells and consecutive demyelination. Spinal cord tissue emulsified in complete Freund's adjuvant clinical disease in the genetically susceptible Dark Agouti rats (DA) but not in Albino Oxford (AO) rats although similar inflammatory infiltrates in the CNS are observed in both strains 10-12 days after induction. We have shown that the resistance to clinical disease of AO rats is associated with rapid clearance of infiltrating mononuclear cells by a mechanism of apoptosis. Here, we demonstrate by immunohistochemical and FACS analyses of the expression of CD11b/c that microglial cells respond differently to disease induction in the two strains. Whereas microglial cells are activated throughout the period of day 10-28 days after EAE induction in AO rats they are only activated at the inception and resolution phases but not at the peak of clinical disease in DA rats when there is the highest level of CD4+ T cell infiltration. Our findings are compatible with the notion that microglia terminate effector T cells by apoptosis and that lack of this mechanism as evidenced by the lack of CD11b/c expression, support T cell survival and clinical expression of disease.     

Nonencephalitogenic CD4-CD8- V alpha 2V beta 8.2+ anti-myelin basic protein rat T lymphocytes inhibit disease induction.

Recently there has been a number of reports suggesting that CD4-CD8- T cells participate in the processes of inflammatory reaction. In an attempt to delineate the distinctive functions of double negative (DN) T lymphocytes in an autoimmune-induced disease, we isolated and cloned such T cells, along with control CD4+ cells, from Lewis rats immunized with guinea-pig myelin basic protein in CFA. Both clones proliferated in response to the guinea-pig myelin basic protein and its synthetic encephalitogenic peptide, and expressed the same TCR V genes homologous to the mouse V alpha 2 and V beta 8.2 families that appear to be the defining entity of experimental autoimmune encephalomyeltis (EAE). Moreover, the TCR D and J region gene products of the DN cell were found to be similar to another encephalitogenic rat T cell clone. The two T clones did not differ markedly in their ability to produce TNF and IL-2 and to adhere to vascular wall-derived extracellular matrix- and laminin-coated plates. Surprising, therefore, was the finding that, although the CD4+ T lymphocytes were capable of inducing EAE, the DN cells did not elicit disease but rather inhibited subsequent EAE induction. Thus, TCR V alpha 2V beta 8.2 and its junctional region gene products are not the only prerequisite segment for a T cell to become encephalitogenic. We suggest that the important determinants of the T cell ability to induce disease are features of the T cell, other than or in addition to, the T cell receptor.     

Characterization of the antigen specificity and TCR repertoire,and TCR-based DNA vaccine therapy in myelin basic protein-induced autoimmune encephalomyelitis in DA rats

Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62-75 sequence (MBP(62-75)) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP(62-75) was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vbeta10 and Vbeta15 spectratype expansion was always found in MBP(62-75)-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vbeta10- and Vbeta15-positive T cells. First, the adoptive transfer experiments revealed that Vbeta10-positive T line cells derived from MBP(62-75)-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vbeta10 and Vbeta15, alone or in combination, ameliorated MBP(62-75)-induced EAE. Collectively, it was strongly suggested that Vbeta10- and Vbeta15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.     

Functional heterogeneity among CD4+ encephalitogenic T cells in recruitment of CD8+ T cells in experimental autoimmune encephalomyelitis

Inoculation of Lewis rats with live or attenuated (irradiated or paraformaldehyde-fixed) CD4+ encephalitogenic T cells (S1 line) protects the recipients from transferred experimental autoimmune encephalomyelitis (tEAE) induced by S1 cells. A CD8+ T lymphocyte population specifically activated against the EAE-inducing S1 cells can be readily isolated from the lymphoid organs of pretreated animals. We show, in the present study, that encephalitogenic T cell lines derived from Lewis rats differ in their ability to induce resistance against tEAE in vivo and to stimulate CD8+ cell proliferation in vitro. We also demonstrate that the S19 line of encephalitogenic T cells, in combination with myelin basic protein (MBP), can stimulate CD8+ cell proliferation in vitro. The CD8+ cells generated in this way strongly suppress MBP-specific T cell proliferation in vitro. This combined effect of T cells and MBP was also evident in vivo. Neither S19 cells nor MBP alone induced resistance against S19-mediated tEAE, rather coinjection of these cells and MBP was required. Our results suggest that resistance to EAE is mediated by distinct populations of encephalitogenic T cells that activate Ts cells through different mechanisms. In some instances, both autoreactive T cells and their relevant autoantigen(s) may be needed to activate Ts cells in vivo.     

Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8+ T cells in vivo.

CD8+ T cells from TCR transgenic 2C mice, specific for SIYRYYGL peptide bound to H-2Kb, were adoptively transferred into C57BL/6 recipients to allow monitoring of their location, numbers, and phenotype upon peptide challenge. Recipients were primed by s.c. injection of SIYRYYGL alone or with CFA or IL-12, and the transferred cells then tracked by flow cytometry using the 1B2 mAb specific for the 2C TCR. Peptide alone induced a transient and weak expansion of 1B2+ cells in the draining lymph nodes (DLN) by day 3, but these cells were tolerant to secondary peptide challenge. In contrast, priming with CFA/peptide resulted in a large clonal expansion of 1B2+ cells in DLN by day 3, and the cells exhibited a CD25highCD44high phenotype, blast transformation, and lytic effector function. By day 5, 1B2+ cell numbers decreased in the DLN and increased in the spleen and blood. 1B2+ cells with a memory phenotype persisted through day 60 in the DLN, spleen, and blood and responded to secondary peptide challenge. Immunization with peptide, along with IL-12, mimicked the adjuvant effects of CFA with respect to phenotype, clonal expansion, effector function, and establishment of memory. IL-12 was not unique in providing this adjuvant effect however, since CFA/peptide immunization of IL-12-deficient recipient mice also resulted in 1B2+ T cell activation and clonal expansion. Thus, CFA or IL-12 can enhance Ag-specific CD8+ T cell responses to peptide, demonstrating that an inflammatory cytokine(s) can support activation and prevent tolerance induction.     

CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1-mediated demyelinating disease of the CNS that serves as a model for multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific and Ag-nonspecific T lymphocytes into the CNS. In the present report, we investigated the role of the CXC chemokine CXCL10 (IFN-gamma-inducible protein-10) in the pathogenesis of EAE. Production of CXCL10 in the CNS correlated with the development of clinical disease. Administration of anti-CXCL10 decreased clinical and histological disease incidence, severity, as well as infiltration of mononuclear cells into the CNS. Anti-CXCL10 specifically decreased the accumulation of encephalitogenic PLP(139-151) Ag-specific CD4+ T cells in the CNS compared with control-treated animals. Anti-CXCL10 administration did not affect the activation of encephalitogenic T cells as measured by Ag-specific proliferation and the ability to adoptively transfer EAE. These results demonstrate an important role for the CXC chemokine CXCL10 in the recruitment and accumulation of inflammatory mononuclear cells during the pathogenesis of EAE.     

Influence of mucosal adjuvants on antigen passage and CD4+ T cell activation during the primary response to airborne allergen

Ag delivery via the nasal route typically induces tolerance or fails to polarize CD4+ T cell responses unless an adjuvant is provided. To better understand this process, we assessed the effects of two mucosal adjuvants, Escherichia coli LPS and cholera toxin (CT), on Ag passage and T cell activation in the draining lymph nodes (DLN) of BALB/c mice following per nasal administration of the model protein allergen, OVA. We found a range of cell types acquired small amounts of fluorescent OVA in the DLN 4 h after per nasal administration. However, this early uptake was eclipsed by a wave of OVA+CD8alpha(low) dendritic cells that accumulated in the DLN over the next 20 h to become the dominant OVA-processing and -presenting population. Both LPS and CT stimulated increases in CD80 and CD86 expression on OVA+CD8alpha(low) DC. LPS also increased the number of OVA+CD8alpha(low) dendritic cells accumulating in the DLN. When the primary T cell response was examined after adoptive transfer of CD4+ T cells from DO11.10 mice, CT and LPS stimulated surprisingly similar effects on T cell activation and proliferation, IL-4 and IFN-gamma priming, and memory T cell production. Despite these similarities, T cell recipients immunized with CT, but not LPS, developed lung eosinophilia upon secondary OVA challenge. Thus, we found no bias within the DLN in Ag handling or the primary T cell response associated with the eventual Th2 polarization induced by CT, and suggest that additional tissue-specific factors influence the development of allergic disease in the airways.     

In vivo immunomodulation by monoclonal anti-CD4 antibody. II. Effect on T cell response to myelin basic protein and experimental allergic encephalomyelitis

In vivo administration of anti-CD4 mAb (GK1.5) has been shown to be effective in preventing acute and relapsing experimental allergic encephalomyelitis (EAE). In the present report we have studied the depletion of CD4+ cells by a single dose of GK1.5 on the immune response to myelin basic protein and in the development of EAE. Our studies show that depletion of CD4 cells in mice that had received encephalitogenic CD4+ T cells altered the kinetics of acute and relapsing EAE, but did not prevent disease altogether. The in vitro T cell proliferative response to myelin basic protein in lymph node cells was maintained in the presence of significant depletion of CD4+ cells. These studies indicate that the population of Ag-reactive cells to be large and relatively refractory to antibody therapy. The implication of these results to therapy of human autoimmune disease is discussed.     

Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35-55 from myelin oligodendrocyte glycoprotein (pMOG(35-55)) consistently activate a high proportion of CD8+ alphabetaTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG(35-55). CNS lesions in pMOG(35-55) CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1(-/-) mice, but not in isogeneic beta2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1(-/-) mice in which disease was induced adoptively with <1 x 10(6) T cells sensitized to pMOG(35-55). It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.     

Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG_35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2^−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2^−/− mice had significantly higher number of CD4⁺ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2^−/− primed lymphocytes induced clinical signs of the disease in ST2^−/− as well as in WT mice. MOG_35–55 restimulated ST2^−/− CD4⁺ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4⁺ cells. ST2^−/− mice had higher percentages of CD4⁺ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4⁺ cells. Draining lymph nodes of ST2^−/− mice contained higher percentage of CD11c⁺CD11b⁺CD8⁻ cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2^−/− but not WT dendritic cells induced EAE in MOG_35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.     

Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.     

CD4+ suppressor cells differentially affect the production of IFN-gamma by effector cells of experimental autoimmune encephalomyelitis

Spleen cells from rats that have recovered from experimental autoimmune encephalomyelitis (EAE) suppress the production of IFN-gamma by effector T cells of EAE in an Ag-specific manner. These postrecovery suppressor cells also inhibit EAE in vivo. Fractionation of the postrecovery suppressor spleen cells on nylon wool and OX-8 coated plates yields a nylon wool-adherent CD4+ suppressor cell population that, when cocultured with effector T cells, suppresses IFN-gamma production by these effector cells. In contrast, the nylon wool-adherent, CD4+ postrecovery suppressor cell population fails to inhibit the production of IL-2 by the effector T cells. In further experiments, the effector T cell population was depleted of CD8+ cells and cocultured with the nylon wool-adherent, CD4+ postrecovery suppressor cells, and the supernatants were assayed for IFN-gamma and IL-2. IFN-gamma production was inhibited in these cultures but IL-2 production was not inhibited. Irradiated effector T cells were cocultured with CD4+ postrecovery suppressor cells, without myelin basic protein, in an effort to determine whether the mechanism of differential lymphokine suppression involved an anti-idiotypic response against effector T cells. No IL-2 was produced, indicating that there was no CD4+ suppressor cell mediated anti-idiotypic response against effector T cells. These studies suggest that the suppressor cell is a nylon wool adherent, CD4+ T cell that functions to down-regulate EAE effector T cells by differential inhibition of lymphokine production.     

CD5-CK2 binding/activation-deficient mice are resistant to experimental autoimmune encephalomyelitis: protection is associated with diminished populations of IL-17-expressing T cells in the central nervous system

Regulating the differentiation and persistence of encephalitogenic T cells is critical for the development of experimental autoimmune encephalomyelitis (EAE). We reported recently that CD5 has an engagement-dependent prosurvival activity in T cells that played a direct role in the induction and progression EAE. We predicted that CD5 regulates T cell apoptosis/survival through the activation of CK2, a prosurvival serine/threonine kinase that associates with the receptor. To test this hypothesis, we generated mice expressing CD5 with the inability to bind and activate CK2 and assessed their susceptibility to EAE. We found mice deficient in CD5-CK2 signaling pathway were mostly resistant to the development of EAE. Resistance to EAE was associated with a dramatic decrease in a population of effector infiltrating Th cells that coexpress IFN-gamma and IL-17 and, to a lesser extent, cells that express IFN-gamma or IL-17 in draining lymph nodes and spinal cords. We further show that T cells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation; however, following restimulation, they rapidly develop nonresponsiveness and exhibit elevated activation-induced cell death. Our results provide a direct role for CD5-CK2 pathway in T cell activation and persistence of effector T cells in neuroinflammatory disease. This study predicts that targeting of IFN-gamma(+)/IL-17(+) infiltrating Th cells will be useful for the treatment of multiple sclerosis and other systemic autoimmune diseases.     

HgCl2-induced perturbation of the T cell network in experimental allergic encephalomyelitis. II. In vivo demonstration of the role of T suppressor and contrasuppressor cells.

In the companion paper (J. Rossert et al., Cell. Immunol. 137, 1991), we showed by using limiting dilution analysis that Lewis (LEW) rats injected with HgCl2 and immunized with myelin (LEWHg/MYE) exhibit anti-basic protein CD4+ T helper cells (Th), at least 10-fold more frequent CD8+ T suppressor cells (Ts), and T contrasuppressor cells (Tcs). These Tcs cells were shown to be CD4+ T cells adhering to Vicia villosa (VV) lectin and allowed Th cells to proliferate despite the presence of Ts cells. The CD8+ Ts cells might be responsible for the protection from experimental allergic encephalomyelitis (EAE) observed in about 70% of LEW rats injected with HgCl2. The concomitant presence of CD4+ Tcs cells might explain that 30% of the rats escaped this protection. The aim of this work is to demonstrate in vivo the roles of CD8+ Ts cells and Tcs cells in mercury-induced protection from EAE. It will be shown that LEWHg/MYE rats depleted of CD8+ cells as well as LEWHg/MYE rats transferred with VV lectin-adherent Tcs cells develop EAE. These data demonstrate that CD8+ Ts cells are responsible for HgCl2-induced protection and that Tcs cells are involved in the control of Ts cells in vivo.     

IFN-gamma shapes immune invasion of the central nervous system via regulation of chemokines

Dynamic interplay between cytokines and chemokines directs trafficking of leukocyte subpopulations to tissues in autoimmune inflammation. We have examined the role of IFN-gamma in directing chemokine production and leukocyte infiltration to the CNS in experimental autoimmune encephalomyelitis (EAE). BALB/c and C57BL/6 mice are resistant to induction of EAE by immunization with myelin basic protein. However, IFN-gamma-deficient (BALB/c) and IFN-gammaR-deficient (C57BL/6) mice developed rapidly progressing lethal disease. Widespread demyelination and disseminated leukocytic infiltration of spinal cord were seen, unlike the focal perivascular infiltrates in SJL/J mice. Gr-1+ neutrophils predominated in CNS, and CD4+ T cells with an activated (CD69+, CD25+) phenotype and eosinophils were also present. RANTES and macrophage chemoattractant protein-1, normally up-regulated in EAE, were undetectable in IFN-gamma- and IFN-gammaR-deficient mice. Macrophage inflammatory protein-2 and T cell activation gene-3, both neutrophil-attracting chemokines, were strongly up-regulated. There was no induction of the Th2 cytokines, IL-4, IL-10, or IL-13. RNase protection assays and RT-PCR showed the prevalence of IL-2, IL-3, and IL-15, but no increase in IL-12p40 mRNA levels in IFN-gamma- or IFN-gammaR-deficient mice with EAE. Lymph node cells from IFN-gamma-deficient mice proliferated in response to myelin basic protein, whereas BALB/c lymph node cells did not. These findings show a regulatory role for IFN-gamma in EAE, acting on T cell proliferation and directing chemokine production, with profound implications for the onset and progression of disease.     

CD24 on the resident cells of the central nervous system enhances experimental autoimmune encephalomyelitis

CD24 is a cell surface glycoprotein that is expressed on both immune cells and cells of the CNS. We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model for the human disease multiple sclerosis (MS). The development of EAE requires CD24 expression on both T cells and non-T host cells in the CNS. To understand the role of CD24 on the resident cells in the CNS during EAE development, we created CD24 bone marrow chimeras and transgenic mice in which CD24 expression was under the control of a glial fibrillary acidic protein promotor (AstroCD24TG mice). We showed that mice lacking CD24 expression on the CNS resident cells developed a mild form of EAE; in contrast, mice with overexpression of CD24 in the CNS developed severe EAE. Compared with nontransgenic mice, the CNS of AstroCD24TG mice had higher expression of cytokine genes such as IL-17 and demyelination-associated marker P8; the CNS of AstroCD24TG mice accumulated higher numbers of Th17 and total CD4+ T cells, whereas CD4+ T cells underwent more proliferation during EAE development. Expression of CD24 in CD24-deficient astrocytes also enhanced their costimulatory activity to myelin oligodendrocyte glycoprotein-specific, TCR-transgenic 2D2 T cells. Thus, CD24 on the resident cells in the CNS enhances EAE development via costimulation of encephalitogenic T cells. Because CD24 is increased drastically on resident cells in the CNS during EAE, our data have important implications for CD24-targeted therapy of MS.     

LF 15-0195 inhibits the development of rat central nervous system autoimmunity by inducing long-lasting tolerance in autoreactive CD4 T cells

Experimental autoimmune encephalomyelitis (EAE) is a T cell-dependent autoimmune disease induced in susceptible animals by a single immunization with myelin basic protein (MBP). LF 15-0195 is a novel immunosuppressor that has been shown to have a potent immunosuppressive effect in several pathological manifestations. The purpose of this study was to investigate the effect of this drug on the induction and progression of established rat EAE and to dissect the mechanisms involved. We show that LF 15-0195 administration at the time of MBP immunization reduces the incidence and severity of EAE in Lewis rats. This drug also inhibits ongoing and passively induced EAE, indicating that LF 15-0195 affects already differentiated pathogenic lymphocytes. Compared with lymph node cells from untreated rats, lymphocytes from MBP-immunized rats treated with LF 15-0195 proliferated equally well in response to MBP in vitro, while their ability to produce effector cytokines and to transfer EAE into syngeneic recipients was significantly reduced. This phenomenon is stable and long-lasting. Indeed, neither IL-12 nor repeated stimulation with naive APC and MBP in vitro rendered MBP-specific CD4 T cells from protected rats encephalitogenic. In conclusion, LF 15-0195 treatment suppresses EAE by interfering with both the differentiation and effector functions of autoantigen-specific CD4 T cells.     

Cytokine- and Ig-producing T cells in mucosal effector tissues: analysis of IL-5- and IFN-gamma-producing T cells, T cell receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues.

The present study has focused on the analysis of cytokine- and Ig-producing mononuclear cells (MC) that reside in the salivary glands and their associated tissues (SGAT) in the oral region. The SGAT are located under the mandibular area and consist of submandibular glands, periglandular lymph nodes, and cervical lymph nodes. MC were isolated from individual SGAT and examined for T cell subsets and TCR expression, in comparison with T cells obtained from other mucosa-associated and systemic tissues. Forty to fifty percent of MC in submandibular glands were CD3+ T cells, equally divided into CD4+ CD8- and CD4- CD8+ T cell subsets. On the other hand, the intestinal lamina propria and Peyer's patches possessed a approximately 2 to 3:1 ratio of CD4+ CD8- to CD4- T cells. A high frequency of CD4- CD8- (double negative) (DN) T cells (approximately 6 to 10%) was also isolated from submandibular glands. In contrast, approximately 70 to 90% of MC in periglandular lymph nodes and cervical lymph nodes were CD3+ T cells and like the peripheral lymph nodes consisted of fivefold higher numbers of CD4+ CD8- than CD4- CD8+ T cells, with low numbers of DN cells (less than 5%). When expression of gamma/delta and alpha/beta TCR was examined in individual T cell subsets of submandibular glands, the CD4- CD8+ and DN T cell fractions contained 25% and 100% gamma/delta TCR+ cells, respectively. On the other hand, essentially all CD4+ CD8- T cells in SGAT as well as CD4- CD8+ cells in periglandular lymph nodes and cervical lymph nodes were alpha/beta TCR+ T cells. When cytokine production was examined by using IFN-gamma- and IL-5-specific enzyme-linked immunospot assays, the CD3+ CD4+ CD8- T cells in submandibular glands contained T cells spontaneously producing IFN-gamma and IL-5. Further, IL-5 spot-forming cells (SFC) were two- to threefold greater in number, compared with IFN-gamma SFC. The periglandular lymph node T cells contained cytokine producing cells with a ratio of 2:1 for IL-5 and IFN-gamma SFC cells, whereas cervical lymph node T cells did not produce cytokines unless stimulated with T cell mitogens. When the isotype distribution of Ig-producing cells was examined among SGAT, submandibular glands contained large numbers of IgA-producing cells, with few IgM- and IgG-producing cells, a pattern similar to that of the lamina propria. Further, elevated numbers of IgA-secreting cells were also seen in periglandular lymph nodes but not in cervical lymph nodes.(ABSTRACT TRUNCATED AT 400 WORDS)