Strongyloides ratti: implication of mast cell-mediated expulsion through FcepsilonRI-independent mechanisms.

In order to examine whether FcepsilonRI-dependent degranulation of intestinal mast cells is required for expulsion of intestinal nematode Strongyloides ratti, CD45 exon6-deficient (CD45-/-) mice were inoculated with S. ratti. In CD45-/- mice, egg excretion in feces persisted for more than 30 days following S. ratti larvae inoculation, whereas in wild-type (CD45+/+) mice, the eggs completely disappeared by day 20 post-infection. The number of intestinal mucosal mast cells, which are known effector cells for the expulsion of S. ratti, was 75% lower in CD45-/- mice compared with that in CD45+/+ mice. Adoptive transfer of wild-type T cells from CD45+/+ mice into CD45-/- mice reduced the duration of S. ratti infection to comparable levels observed in CD45+/+ mice, with concomitant increases in intestinal mucosal mast cells. These results showed that CD45 is not involved in the effector function of intestinal mucosal mast cells against S. ratti infection. Since FcepsilonRI-dependent degranulation of mast cells is completely impaired in these CD45 knockout mice, we conclude that FcepsilonRI-dependent degranulation is not required in the protective function of intestinal mucosal mast cells against primary infection of S. ratti.

Strongyloides ratti: the role of interleukin-5 in protection against tissue migrating larvae and intestinal adult worms.

To determine the role of interleukin-5 (IL-5) and eosinophils in protection against Strongyloides ratti, mice treated with anti-IL-5 monoclonal antibody (mAb) were infected with S. ratti larvae. Strongyloides ratti egg numbers in faeces (EPG) in mAb treated mice were higher than those in control mice on days 6 and 7 after inoculation. The numbers of migrating worms in mAb treated mice 36 h after inoculation were higher than those observed in control mice. Intestinal worm numbers in mAb treated mice 5 days after inoculation were higher than those in control mice. These results show that eosinophils effectively protected the host against S. ratti infection by mainly the larval stage in primary infections. The involvement of eosinophils in protection against secondary infection was also examined. Before secondary infection, mice were treated with anti-IL-5 mAb and infected with S. ratti. Patent infections were not observed in either mAb treated or control Ab treated mice. The numbers of migrating worms in the head and lungs of mAb treated mice increased to 60% of that in primary infected mice. Intestinal worms were not found in mAb treated mice or in control mice after oral implantation of adult worms. Eosinophils were therefore mainly involved in protection against tissue migrating worms in secondary infections.

Recombinant Sendai virus vectors for activated T lymphocytes.

T-lymphocyte-directed gene therapy has potential as a treatment of subjects with immunological disorders. One current limitation of this therapeutic strategy is low gene transfer efficiency, even when complex procedures are used. We report herein that a recombinant Sendai virus vector (SeV) was able to overcome this issue. Using jellyfish enhanced green fluorescent protein gene (EGFP), we found that SeV was able to transduce and express a foreign gene specifically and efficiently in activated murine and human T cells, but not in naive T cells, without centrifugation or reagents including polybrene and protamine sulfate; the present findings were in clear contrast to those demonstrated with the use of retroviruses. The transduction was selective in antigen-activated T cells, while antigen-irrelevant T cells were not transduced, even under bystander activation from specific T-cell responses by antigens ex vivo. Receptor saturation studies suggested a possible mechanism of activated T-cell-specific gene transfer, ie, SeV might attach to naive T cells but might be unable to enter their cytoplasm. We therefore propose that the SeV vector system may prove to be a potentially important alternative in the area of T-cell-directed gene therapy used in the clinical setting.

IL-7 prevents both caspase-dependent and -independent pathways that lead to the spontaneous apoptosis of i-IEL.

Intestinal intraepithelial lymphocytes (i-IEL) readily undergo spontaneous apoptosis in vitro through an unclear mechanism. Here we examined the relationship between caspases, which plays a major role in apoptosis, and IL-7 in the spontaneous apoptosis of i-IEL in vitro. We demonstrated that IL-7 and zVAD prevented the spontaneous apoptosis of i-IEL by approximately 50% and 25% respectively with no additive protection seen when both are used. IL-7 preferentially prevented the apoptosis of gammadelta i-IEL, while zVAD equally prevented the apoptosis of gammadelta and alphabeta i-IEL. Lastly, we demonstrated that the spontaneous apoptosis of i-IEL is associated with a marked increase in caspase activity. Caspase activity was completely inhibited by zVAD, but only slightly by IL-7. Overall these results suggest that two pathways lead to the spontaneous apoptosis of i-IEL, one which is caspase dependent and the other which is caspase independent. IL-7 appears to exert its effect on i-IEL undergoing spontaneous by partially inhibiting both apoptotic pathways.

The effect of interleukin-4 on the induction of intestinal mast cells and chronological cytokine profiles during intestinal nematode Strongyloides ratti infection.

The effect of interleukin-4 (IL-4) on the induction of intestinal mast cells and cytokine profiles during Strongyloides ratti infection was studied using IL-4 knockout (IL-4 KO) mice. The antigen-specific proliferative response of mesenteric lymph node cells was not impaired in IL-4 KO mice. The number of intestinal mast cells induced in IL-4 KO mice during S. ratti infection was 2- to 3-fold lower than that observed in WT mice. Intestinal mastocytosis had disappeared in IL-4 KO mice by day 21 postinfection, when significant mastocytosis continued to be observed in WT mice. In mesenteric lymphnode of IL-4 KO, IL-3 production decreased and mice IFN-gamma production significantly increased as compared with those of WT mice. The numbers of eggs excreted per gram of feces (EPG) by IL-4 KO mice were greater than those excreted by WT mice on day 6 postinfection, but no difference was observed in the subsequent period. In conclusion, intestinal mast cells are induced during S. ratti infection in the absence of IL-4, and IL-4 is not essential for protection against intestinal adult worms of S. ratti.

Dietary conjugated linoleic acid increases immunoglobulin productivity of Sprague-Dawley rat spleen lymphocytes.

The dietary effects of conjugated linoleic acid (CLA) on Ig production of Sprague-Dawley rats were examined at various doses such as 0 (control), 0.05, 0.10, 0.25, and 0.50%. CLA increased IgG and IgM production of spleen lymphocytes in a dose-dependent manner, and these levels reached a plateau at 0.25%. IgA production was not detected in the control group, while it was detected in all CLA-fed groups and IgA productivity of spleen lymphocytes increased in a dose-dependent manner at the doses from 0.05 to 0.50%. Dietary CLA did not affect serum Ig levels. The major fatty acid composition of spleen lymphocytes was not affected by dietary CLA, which itself was hardly incorporated into the cells. In an in vitro assay, the effects of CLA and its oxidative derivatives, furan type fatty acids, on Ig productivity were also examined. As a result, 100 microM CLA suppressed Ig production of spleen lymphocytes and the degree was as follows IgA > IgG > IgM. Each CLA isomer and the furan type fatty acids also suppressed Ig production but the degree was weaker than the mixture of CLA isomers. In this result, dietary CLA increased Ig productivity of spleen lymphocytes in vivo.

N-3 polyunsaturated fatty acids accentuate B16 melanoma growth and metastasis through suppression of tumoricidal function of T cells and macrophages.

BACKGROUND: The antitumor effects of the n-3 polyunsaturated fatty acids (PUFAs) are still controversial and as yet undefined. MATERIALS AND METHODS: EPA-28, a fish oil enriched with n-3 PUFAs including eicosapentaenoic and docosahexaenoic acids, was administered subcutaneously into C57BL/6 mice before and after subcutaneous inoculation of B16 melanoma cells. The effects of EPA-28 on the antitumor activities of T cells and macrophages were investigated. RESULTS: The treatment of the mice with EPA-28 before and after the tumor inoculation enhanced the growth and metastasis of B16 melanoma and decreased the survival rate of the tumor-bearing mice. The treatment also decreased the number of CD4+ T cells in the spleen and tumor draining lymph nodes on day 14 after the tumor inoculation. Moreover, EPA-28 suppressed the antimelanoma cytolytic activity of T cells and macrophages of the tumor-bearing mice. CONCLUSION: The results suggest that EPA-28 treatment increased both the growth and metastasis of B16 melanoma cells by suppressing the cytolytic function of both T cells and macrophages.

Development of dendritic epidermal T cells with a skewed diversity of gamma delta TCRs in V delta 1-deficient mice.

One of the most intriguing features of gammadelta T cells that reside in murine epithelia is the association of a specific Vgamma/Vdelta usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the "first wave" Vgamma5+ fetal thymocytes and overwhelmingly express the canonical Vgamma5/Vdelta1-TCRs lacking junctional diversity. Targeted disruption of the Vdelta1 gene resulted in a markedly impaired development of Vgamma5+ fetal thymocytes as precursors of DETCs; however, gammadeltaTCR+ DETCs with a typical dendritic morphology were observed in Vdelta1-/- mice and their cell densities in the epidermis were slightly lower than those in Vdelta1+/- epidermis. Moreover, the Vdelta1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vgamma5+ DETCs were predominant in the Vdelta1-/- epidermis, though Vgamma5- gammadeltaTCR+ DETCs were also detected. The Vgamma5+ DETCs showed a typical dendritic shape, gammadeltaTCR(high), and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vgamma5- gammadeltaTCR+ DETCs showed a less dendritic shape, gammadeltaTCR(low), and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vgamma/Vdelta-chain usage but requires expression of a limited diversity of gammadeltaTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.

Human skin xenograft rejection in CD45 exon-6 knockout mice: the implication of involvement of a direct pathway.

The results of previous studies indicate that only CD4+ T cells generated via the indirect pathway play an essential role in causing discordant skin xenograft rejection. The present study was conducted in an attempt to clarify further the roles of effector T cells generated via direct pathways on discordant xenograft rejection using CD45 exon-6 knockout (CD45-/-; C57BL/6 (B6): H-2b) mice. It has been strongly suggested that CD45 exon-6 knockout mice have profound impairment in T-cell functions via an indirect pathway. When human skin was grafted onto untreated normal C57BL/6 (B6; H-2b) mice, rejection occurred within 12 days; however, in the CD45 exon-6 knockout mice, the grafts lasted for slightly longer as in fully allogeneic C3H (H-2k) skin rejection, with a mean survival time +/- SD of 19.4 +/- 1.5 days and median survival times of 19 days. The difference in survival periods between the human and C3H skin grafts in the CD45 knockout mice was not statistically significant. Both CD4+ and CD8+ T cells seemed activated in the spleens of these CD45 exon-6 knockout mice 10 days after the human skin grafting. These results suggest that effector T cells generated via a direct pathway can cause discordant skin xenograft rejection, and that CD45 exon-6 knockout mice can generate effector T cells via a direct pathway to reject discordant skin xenografts, similarly to fully allogeneic skin allografts.

Characterization of CD4- CD8- CD3+ T-cell receptor-alphabeta+ T cells in murine cytomegalovirus infection.

In this study, we have investigated that after the intraperitoneal infection with murine cytomegalovirus (MCMV), the CD3+ CD4- CD8-(double negative; DN) T-cell receptor (TCR)alphabeta+ T cells increased in peritoneal cavity, liver and spleen in both resistant C57BL/6 and susceptible BALB/c mice. The total cellular population of these cells showed peak levels around day 5 after infection in all the three investigated organs and the following phenotypical and functional characteristics emerged. The peritoneal DN TCRalphabeta+ T cells expressed highly skewed TCRVbeta8 on day 5 after infection compared with the uninfected mice, but those in spleen and liver showed moderate and low skewed TCRVbeta8, respectively. The percentages of NK1.1+ DN TCRalphabeta+ T cells gradually decreased as did modulation of some of their activation markers consistent with an activated cell phenotype. The peritoneal DN TCRalphabeta+ T cells on day 5 after infection expressed the genes of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha, Eta-1 (early T-cell activation-1) and MCP-1 (monocyte chemoattractant protein 1) but lacked expression of interleukin-4 (IL-4). After in vitro stimulation with phorbol 12-myristate 13-acetate and calcium ionophore in the presence of Brefeldin A, higher frequencies of intracellular IFN-gamma+ DN TCRalphabeta+ T cells were detected in all three investigated organs of infected mice compared with those of uninfected mice. Stimulation of peritoneal DN TCRalphabeta+ T cells with plate-bound anti-TCRbeta monoclonal antibodies showed proliferation and also produced IFN-gamma but not IL-4. These results suggest that DN TCRalphabeta+ T cells were activated and may have an antiviral effect through producing IFN-gamma and some macrophage-activating factors during an early phase of MCMV infection.

Increased urinary nitrate excretion associated with hepatic allograft rejection in experimental rat models and clinical cases.

In this study, we investigated the relationship between excretion of urinary nitrate as a stable end-product of nitric oxide (NO) metabolism and hepatic allograft rejection. In experimental rat models, hepatic allograft rejection was associated with increased nitrate excretion with a peak on postoperative day 5. The severity of the hepatic allograft rejection was dependent on the increased urinary nitrate excretion. No significant increase in urinary nitrate excretion was observed in cases in which effective immunosuppression was achieved. Inducible nitric oxide synthase mRNA expression was upregulated parallel to interferon-gamma gene expression in the graft-infiltrating mononuclear cells and spleen cells from the recipients. In clinical cases, urinary nitrate excretion increased parallel to increased serum cytosolic enzymes that accompanied rejection. These results suggest that urinary nitrate excretion is a useful indicator for the surveillance of graft rejection and the monitoring of therapeutic effects of antirejection treatments.

beta-estradiol suppresses T cell-mediated delayed-type hypersensitivity through suppression of antigen-presenting cell function and Th1 induction.

BACKGROUND: Although an immunomodulatory role for estrogens has long been demonstrated by experimental and clinical observations, the mechanism by which estrogens exert their effect on T cells has not been clearly defined. METHODS: In this study we analyzed the effects of beta-estradiol (E2), at its contraceptive dose, on the delayed-type hypersensitivity (DTH) to purified protein derivatives (PPD) and associated immune response in female mice. RESULTS: E2 treatment decreased PPD-specific DTH response, which coincided with a decrease in the leukocytes numbers in the draining lymph nodes (DLN) and spleen compared with control mice. E2 treatment also suppressed the in vitro PPD-specific proliferative response of DLN and spleen cells from PPD-primed mice. The analysis of production and gene expression of cytokines by DLN cells demonstrated that E2 treatment suppressed IL-2 and IFN-gamma production in response to PPD in vitro. In contrast, IL-4 and IL-10 gene expression by DLN cells of E2-treated mice, taken 24 h after in vivo restimulation of mice with PPD, was enhanced. Furthermore, we found that spleen APC from E2-treated mice failed to induce optimum proliferation of the PPD-primed T cells in response to PPD in vitro. The impaired APC function by E2 was not due to induction of suppressor cell activity because addition of the normal spleen APC to APC from E2-treated mice restored the proliferative response of the PPD-primed T cells in response to PPD. CONCLUSION: Our results suggest that the E2-mediated inhibition of DTH reaction is due to a combination of the down regulation of APC function and deviation of the immune response from Th1-type to Th2-type.

The crucial role of granulocytes in the early host defense against Strongyloides ratti infection in mice.

The involvement of granulocytes in the host early defense against the nematode, Strongyloides ratti, was studied. It was confirmed that granulocytes were effectively depleted for 4 days by anti-granulocyte monoclonal antibody (anti-Gr-1). To examine the involvement of granulocytes in the host defense against migrating larvae, 2,000 S. ratti infective larvae (L3) were inoculated subcutaneously 1 day after antibody treatment. The number of S. ratti eggs secreted in feces (EPG) was higher in the granulocyte-depleted group than in the control group. The number of migrating larvae also increased in the granulocyte-depleted group in accordance with the increase in EPG. Therefore granulocytes are crucial for the host early defense against migrating larvae of S. ratti. Next, the involvement of granulocytes in the intestinal early defense was examined. Mice were treated with the antibody on day 3 post-infection. On that day, almost all inoculated larvae reached the intestine and molted to become adults. EPG on day 5 post-infection was increased by the antibody treatment, but no effect was observed on intestinal worm numbers. The fecundity (EPG/worm number) of S. ratti adult worms in the granulocyte-depleted group was higher than that in the control group. Thus granulocytes are also involved in the intestinal early defense through suppressing fecundity of the adult worms. On the other hand, the depletion of granulocytes had no effect on the late adaptive response against S. ratti adult worms (e.g. number of intestinal mucosal mast cells, time of worm expulsion). These results suggest that granulocytes are mainly involved in the host early defense against parasites.

Accumulation of common T cell clonotypes in the salivary glands of patients with human T lymphotropic virus type I-associated and idiopathic Sjögren's syndrome.

To clarify the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated Sjögren's syndrome (SS), the TCR Vbeta gene usage by the infiltrating lymphocytes in the target organ was examined. The Vbeta families predominantly used in the labial salivary gland (LSG) from the HTLV-I-seropositive (HTLV-I+) SS patients were more restricted than those from the HTLV-I-seronegative (idiopathic) SS patients, and were commonly Vbeta5.2, Vbeta6, and Vbeta7. The single-strand conformation polymorphism analysis revealed that T cell clonotypes with Vbeta5.2, Vbeta6, and Vbeta7 accumulate in the LSG from the HTLV-I+ and idiopathic SS patients. Among junctional sequences of the most dominant Vbeta7 transcripts, the conserved amino acid motif (QDXG: X is any amino acid) was found in six of the five HTLV-I+ SS patients and was also detected in two of the five idiopathic SS patients. Using the probes specific to the motif, the Vbeta7 transcripts with the motif were detected in the LSG from all of the seven HTLV-I+ and five of the six idiopathic SS patients, but not from eight healthy subjects. The Vbeta7 transcripts with this motif were also detected in the HTLV-I-infected T cell lines obtained from the LSG of an HTLV-I+ SS patient. The accumulation of HTLV-I-infected T cells expressing TCR with the conserved motif was thus indicated. These T cells were commonly present in patients with idiopathic SS and are strongly suggested to most likely be involved in the pathogenesis of both HTLV-I-associated and idiopathic SS.

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection.

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.

Approach to withdrawal from tacrolimus in a fully allogeneic murine skin graft model.

With few exceptions, transplant patients must take immunosuppressants throughout their lives. In this study, we used anti-T-cell receptor (TCR/CD3) monoclonal antibodies (mAbs) to induce immunological tolerance to alloantigens after withdrawal from tacrolimus in a fully allogeneic murine skin graft model. Skin grafts from AKR donor mice were maintained in C57BL/6 recipients by administering tacrolimus for one month. Anti-T-cell receptor (TCR) alphabeta mAb was administered to recipient mice on the day of withdrawal from tacrolimus administration. Seven days after mAb administration, the recipient mice were treated with various combinations of the following treatments: low-dose whole body irradiation, AKR bone marrow transfer (BMT), and anti-CD3 mAb administration. The control recipient mice did not receive treatment with either mAb, nor any other treatment. All the control recipient mice showed rejection of AKR skin grafts 42 days after tacrolimus withdrawal (mean skin graft survival: 77 days). Mice treated with a combination of anti-TCR alphabeta antibody, low-dose irradiation and AKR BMT showed stable chimerism in their peripheral blood lymphocytes and significantly prolonged skin graft survival (mean skin graft survival: >151.2+/-15.3 days). Mice given the combination of anti-TCR alphabeta mAb, anti-CD3 mAb, low-dose irradiation, and AKR BMT exhibited more stable chimerism but had earlier skin graft rejection (mean skin graft survival: 116.7+/-17.6 days) than the mice that did not receive anti-CD3 mAb. These results suggest that anti-TCR alphabeta mAb, but not anti-CD3 mAb, in combination with low-dose irradiation and BMT, is useful for long-lasting allograft survival after withdrawal from tacrolimus in mice with fully allogeneic skin grafts.

Transcriptional regulation of a receptor protein tyrosine phosphatase gene hPTP-J by PKC-mediated signaling pathways in Jurkat and Molt-4 T lymphoma cells.

The recently cloned type II receptor protein tyrosine phosphatase (RPTP) gene hPTP-J is a new member of the MAM (meprin, A5, PTPmicro) domain subfamily. We previously reported that hPTP-J mRNA was detected significantly in Jurkat T lymphoma cells and its expression was completely down-regulated by phorbol myristate acetate (PMA). In this study, we investigated what signaling pathways/molecules are involved in the transcriptional regulation of hPTP-J expression in Jurkat and Molt-4 T cell lines. The hPTP-J transcription was transiently up-regulated 20 min after the addition of PMA (20 ng/ml) to the Jurkat culture, followed by the complete down-regulation in 8 h after PMA addition. The transient up-regulation and the complete down-regulation induced by PMA was blocked by a PKC-specific inhibitor, GF109203X, suggesting that the regulatory effect of PMA on the hPTP-J transcription depends on protein kinase C activation. hPTP-J transcription was down-regulated not only by PMA but also by several signaling modulators including 1-oleoyl-2-acetylglycerol, forskolin, orthovanadate, manumycin and okadaic acid. Therefore, several signaling molecules such as protein tyrosine phosphatases, PP2A/CaMKIV and Ras are required for hPTP-J transcription in Jurkat and Molt-4 cells.

T-Cell hyporesponsiveness induced by activated macrophages through nitric oxide production in mice infected with Mycobacterium tuberculosis.

In active tuberculosis, T-cell response to Mycobacterium tuberculosis is known to be reduced. In the course of Mycobacterium tuberculosis infection in mice, we observed that T-cell proliferation in response to M. tuberculosis purified protein derivative (PPD) reached the maximum level on day 7, then declined to the minimal level on day 14, and persisted at a low level through day 28 postinfection. The frequency of PPD-specific CD4 T cells in the spleen on day 28 decreased to one-sixth on day 7. To further investigate the mechanism of this T-cell hyporesponsiveness, we next analyzed the suppressive activity of spleen macrophages on T-cell function. The nonspecific proliferative response of naive T cells and the PPD-specific proliferative response of T cells were suppressed by day 28 macrophages, but not by day 7 macrophages or naive macrophages. This reduction of proliferative response was restored by addition of nitric oxide synthesis inhibitor, NG-monoethyl-L-arginine monoacetate, but not by monoclonal antibody against interleukin 10 or transforming growth factor beta. These data indicate that the macrophages from mice chronically infected with M. tuberculosis suppress T-cell response through production of nitric oxide, suggesting that nitric oxide-induced elimination mediated by activated macrophages may reduce the T-cell response and the number of mycobacterium-specific CD4 T cells in vivo.