Prakash Nagarkatti

The precise role of cannabinoid receptors (CB)1 and CB2, as well as endogenous ligands for these receptors, on immune cells remains unclear. In the current study, we examined the effect of endogenous and exogenous cannabinoids on murine bone marrow-derived dendritic cells (DCs). Addition of Delta(9)-tetrahydrocannabinol (THC), a major psychoactive component found in marijuana or anandamide, an endogenous cannabinoid, to DC cultures induced apoptosis in DCs. DCs expressed CB1 and CB2 receptors and the engagement of both receptors was necessary to trigger apoptosis. Treatment with THC induced caspase-2, -8, and -9 activation, cleavage of Bid, decreased mitochondrial membrane potential, and cytochrome c release, suggesting involvement of death-receptor and mitochondrial pathways. DCs from Bid-knockout mice were sensitive to THC-induced apoptosis thereby suggesting that Bid was dispensable. There was no induction of p44/p42 MAPK, p38 MAPK, or stress-activated protein/JNK pathway in THC-treated DCs. However, THC treatment induced phosphorylation of IkappaB-alpha, and enhanced the transcription of several apoptotic genes regulated by NF-kappaB. Moreover, inhibition of NF-kappaB was able to block THC-induced apoptosis in DCs. Lastly, in vivo treatment of mice with THC caused depletion of splenic DCs. Together, our study demonstrates for the first time that endogenous and exogenous cannabinoids may suppress the immune response through their ability to induce apoptosis in DCs.

The molecular events that link NADPH oxidase activation and the induction of Toll-like receptor (TLR)-4 recruitment into hepatic lipid rafts in nonalcoholic steatohepatitis (NASH) are unclear. We hypothesized that in liver, NADPH oxidase activation is key in TLR4 recruitment into lipid rafts, which in turn up-regulates NF-κB translocation to the nucleus and subsequent DNA binding, leading to NASH progression. Results from confocal microscopy showed that liver from murine and human NASH had NADPH oxidase activation, which led to the formation of highly reactive peroxynitrite, as shown by 3-nitrotyrosine formation in diseased liver. Expression and recruitment of TLR4 into the lipid rafts were significantly greater in rodent and human NASH. The described phenomenon was NADPH oxidase, p47phox, and peroxynitrite dependent, as liver from p47phox-deficient mice and from mice treated with a peroxynitrite decomposition catalyst [iron(III) tetrakis(p-sulfonatophenyl)porphyrin] or a peroxynitr...

American ginseng is capable of ameliorating cardiac dysfunction and activating Nrf2, a master regulator of antioxidant defense, in the heart. This study was designed to isolate compounds from American ginseng and to determine those responsible for the Nrf2-mediated resolution of inflamed macrophage-induced cardiomyocyte hypertrophy. A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. A bioassay-based fractionization of American ginseng was performed to identify the putative substances which could activate Nrf2-mediated suppression of pro-inflammatory cytokine expression in macrophages and macrophage-mediated pro-hypertrophic growth in cardiomyocytes. A hexane fraction of an anti-inflammatory crude extract of American ginseng was found to be most effective in suppressing the inflammatory responses in macrophages. Preparative, reverse-phase HPLC and a comparative analysis by analytical ...

CD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix (ECM). Recent studies have demonstrated that activation through CD44 leads to induction of effector function in T cells and macrophages. At sites of chronic inflammation as seen in certain infections, autoimmune diseases, allograft rejection, graft-versus-host (GVH) disease and treatment of cancer patients with high doses of interleukin-2, significant damage to the endothelial cells has been known to occur, which leads to the toxicity or pathogenesis associated with the disease. The exact mechanism of endothelial cell damage is not clear, although, it has been widely speculated that immune cells may play a critical role. Studies from our laboratory have used interleukin-2 (IL-2) induced vascular leak syndrome (VLS) as a model to investigate the role of cytolytic lymphocytes in the direct cytotoxicity of endothelial c...

MDSCs are potent immunosuppressive cells that are induced during inflammatory responses, as well as by cancers, to evade the anti-tumor immunity. We recently demonstrated that marijuana cannabinoids are potent inducers of MDSCs. In the current study, we investigated the epigenetic mechanisms through which THC, an exogenous cannabinoid, induces MDSCs and compared such MDSCs with the naïve MDSCs found in BM of BL6 (WT) mice. Administration of THC into WT mice caused increased methylation at the promoter region of DNMT3a and DNMT3b in THC-induced MDSCs, which correlated with reduced expression of DNMT3a and DNMT3b. Furthermore, promoter region methylation was decreased at Arg1 and STAT3 in THC-induced MDSCs, and consequently, such MDSCs expressed higher levels of Arg1 and STAT3. In addition, THC-induced MDSCs secreted elevated levels of S100A8, a calcium-binding protein associated with accumulation of MDSCs in cancer models. Neutralization of S100A8 by use of anti-S100A8 (8H150) in viv...

Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily th...

Exposure to Staphylococcal enterotoxin B (SEB) causes food poisoning, acute inflammatory lung injury, toxic shock syndrome, and often death. In this study, we investigated whether microRNA (miRNA) play a role in regulating SEB-driven inflammation in the lungs. Exposure to SEB caused immune cell infiltration, robust cytokine and chemokine production, compromised lung function, and 100% mortality in mice. We assessed miRNA and mRNA expression in lung infiltrating mononuclear cells following exposure to SEB and found 89 miRNA that were dysregulated (>2-fold) compared with vehicle controls. In silico analysis revealed that the miRNA exhibited biological functions pertaining to cell death and survival, cellular proliferation, and cell cycle progression. Through the use of q-RT PCR, we validated 9 specific miRNA (miR-155, miR-132, miR-31, miR-222, miR-20b, miR-34a, miR-192, miR-193*, and let-7e) and observed that they were predicted to bind the 3'-UTR of a number of genes that were...

Staphylococcal enterotoxin B (SEB) is a potent activator of Vβ8+T-cells resulting in the clonal expansion of ∼30% of the T-cell pool. Consequently, this leads to the release of inflammatory cytokines, toxic shock, and eventually death. In the current study, we investigated if Δ(9) tetrahydrocannabinol (THC), a cannabinoid known for its anti-inflammatory properties, could prevent SEB-induced mortality and alleviate symptoms of toxic shock. We investigated the efficacy of THC against the dual administration (intranasal and i.p.) of SEB into C3H/HeJ mice based on the measurement of SEB-mediated clinical parameters, including cytokine production, cellular infiltration, vascular leak, and airway resistance. In addition, the molecular mechanism of action was elucidated in vitro by the activation of splenocytes with SEB. Exposure to SEB resulted in acute mortality, while THC treatment led to 100% survival of mice. SEB induced the miRNA-17-92 cluster, specifically miRNA-18a, which targeted ...

The impact of the endogenous cannabinoids (AEA, 2-AG, PEA, and virodamine) on the immune cell expressed cannabinoid receptors (CB1, CB2, TRPV-1, and GPR55) and consequent regulation of immune function is an exciting area of research with potential implications in the prevention and treatment of inflammatory and autoimmune diseases. Despite significant advances in understanding the mechanisms through which cannabinoids regulate immune functions, not much is known about the role of endocannabinoids in the pathogenesis or prevention of autoimmune diseases. Inasmuch as CB2 expression on immune cells and its role has been widely reported, the importance of CB1 in immunological disorders has often been overlooked especially because it is not highly expressed on naive immune cells. Therefore, the current review aims at delineating the effect of endocannabinoids on CB1 receptors in T cell driven autoimmune diseases. This review will also highlight some autoimmune diseases in which there is ...

Marijuana is one of the most abused drugs due to its psychotropic effects. Interestingly, it is also used for medicinal purposes. The main psychotropic component in marijuana, Δ(9)-tetrahydrocannabinol (THC), has also been shown to mediate potent anti-inflammatory properties. Whether the immunomodulatory activity of THC is mediated by epigenetic regulation has not been investigated previously. In this study, we employed ChIP-Seq technology to examine the in vivo effect of THC on global histone methylation in lymph node cells of mice immunized with a superantigen, staphylococcal enterotoxin B. We compared genome-wide histone H3 Lys-4, Lys-27, Lys-9, and Lys-36 trimethylation and histone H3 Lys-9 acetylation patterns in such cells exposed to THC or vehicle. Our results showed that THC treatment leads to the association of active histone modification signals to Th2 cytokine genes and suppressive modification signals to Th1 cytokine genes, indicating that such a mechanism may play a cri...

In an attempt to delineate the immunological alterations that may occur following treatment with estrogen, groups of C57BL/6 mice were treated with 75mg/kg body weight of beta-estradiol-17-valerate (E2) or the vehicle. The thymus from these mice were harvested on days 1, 4 and 7 following treatment. The thymocytes from E2-treated mice when cultured in vitro for 24h, showed increased levels of apoptosis when compared to controls. The apoptosis was demonstrable by both TUNEL assay and AnnexinV/propidium iodide (PI) staining. Also, thymic atrophy and increased apoptosis of thymocytes when cultured in vitro were seen when lower doses of E2 (5mg/kg) were administered. The thymus from E2-treated mice on days 4 and 7 also showed a decrease in the percentage of CD4(+)CD8(+) (DP) T cells and an increase in the percentage of CD4(-)CD8(-) (DN), CD4(+) and CD8(+) T cells. However, the total cellularity of all T cell subsets in the thymus was decreased following E2 treatment. Earlier studies fro...

The effect of O-chlorobenzylidene malononitrile (CS) on the immune system was studied in mice given 8 and 16 mg/kg body weight i.p. of the compound, daily for 10 days. The humoral immune response to SRBC was suppressed at both doses, as determined by the antibody producing cells in the spleen. To find out whether the immunosuppression observed was due to the indirect effect of CS on the nutritional status and endocrine balance, serum proteins and corticosterone levels were measured. While the serum proteins did not alter, corticosterone levels rose significantly only in mice receiving higher dose of CS.

Paired serum samples from 25 patients having clinical evidence of arboviral infections and 15 controls were examined for antibodies against dengue, Japanese encephalitis and West nile viruses by haemagglutination inhibition (HI) and indirect fluorescent antibody (IFA) methods. The two tests were comparable in specificity but the IFA test was found to be more sensitive than the HI test. Like the HI test, the antibodies detected by IFA test cross-reacted within the flavivirus group under test. Use of IFA test is recommended to detect flavivirus infections as the test is simple, sensitive and can be carried out rapidly.

The fetus that results from allogeneic mating in an outbred population bears a variety of antigens against which the maternal immune system reacts, but the type of immunity that is elicited by pregnancy does not mediate graft rejection. Previous studies have demonstrated the presence of nonspecific non-thymus derived suppressor cells in the lymph nodes draining the uterus (DLN) and in the uterine decidua during first allogeneic pregnancy. These suppressor cells appear to be small lymphoid cells that inhibit the generation of cytotoxic T cells (CTL) against paternal alloantigens. We now report that after a single allogeneic pregnancy, C3H and A strain mice develop paternal alloantigen-specific suppressor T cells (Thy-1.2+, Lyt-1-2+) that are distributed systemically in peripheral lymph nodes and spleen. These suppressor cells appear to act directly on CTL generation because the frequency of CTL precursors and the ability to produce T helper cells in response to paternal alloantigens ...

The Ca2+ ionophores, A23187 and ionomycin, when used alone at 0.3-5 microM concentrations, were mitogenic to normal resting murine T cells. At these doses, the Ca2+ ionophores inhibited DNA synthesis and growth of a T cell lymphoma and several murine transformed cell lines. Treatment of tumor cells with either ionophore caused a rapid increase in intracellular Ca2+ [( Ca2+]i) followed by a decline and, thereafter, maintenance of a steady level that was slightly above the preactivation levels of [Ca2+]i. Cell cycle analysis revealed that each of the Ca2+ ionophores inhibited tumor cell transition from the G1 phase to the S phase of the cell cycle and, possibly, arrested cells already in the S-phase. These data suggest that an early activation signal such as increased [Ca2+]i may initiate surface to nuclear signals that are different in normal T cells than in transformed cells that may receive opposite effects. Our studies suggest that non-specific activation of tumor cells by various...

We have recently demonstrated that nitrosoureas such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and chlorozotocin (CLZ) can cure almost 100% of mice bearing LSA tumor, syngeneic to C57BL/6 mice. In contrast, similar or higher doses of streptozotocin (STZ) completely failed to cure LSA-bearing mice. Further studies revealed that the efficacy of nitrosoureas may depend on their immunomodulating properties. In the current study, therefore, we investigated the effect of these nitrosoureas on the immune system of normal mice. Treatment of C57BL/6 mice with 5 intraperitoneal injections of 20 mg/kg body weight of BCNU or CLZ caused an increase in the percentage of CD4(-)CD8- T cells and a decrease in the percentage of CD4(+)CD8+ T cells in the thymus. In addition, such treatment also caused an increase in the percentage of CD4+ T cells without significantly affecting the CD8+ T cells in the thymus. However, when total cellularity of the thymus was studied, BCNU and CLZ were found to d...

Recent studies have suggested the existence of two mutually exclusive subpopulations of T helper (Th) cells in the murine immune system, called Th1 which produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 and Th2 which secretes IL-4 and IL-5 but not IL-2. Also, functionally, Th1 cells generally activate the macrophages and mediate delayed-type hypersensitivity whereas Th2 cells provide help efficiently to B cells. In the present study, we investigated the lymphokine secretory properties of two well-characterized autoreactive (self-Ia reactive) T cell clones isolated from normal DBA/2 mice and autoimmune-susceptible MRL-lpr/lpr mice. It was observed that both the autoreactive T cell clones, following activation, produced IL-2, IL-4, and IFN-gamma. They induced hyper-Ia expression and cell proliferation in syngeneic B cells as well as activated the macrophages to exhibit tumoristatic properties. Both clones could also induce T-T network interaction in which syngeneic ...

Aldicarb, a carbamate pesticide used extensively throughout the United States, has been shown in several areas to contaminate drinking water at levels exceeding 100 p.p.b. Recent studies have suggested that aldicarb at levels well below these found in drinking water may lead to alterations in mammalian health. In the present study, we investigated the possible toxic effects of aldicarb on the mammalian immune system. Specifically examined in these studies were the effects of aldicarb on syngeneic mixed lymphocyte reaction (SMLR) in which CD4+ T-helper cells (autoreactive T-cells) respond to self or syngeneic Ia molecules expressed on macrophages. The effect of aldicarb was delineated at both the responder and stimulator cell-level. When C3H mice were injected intraperitoneally with a single dose of 0.1-1000 p.p.b. of aldicarb, it was observed that there was a decrease in the stimulatory functions of macrophages, as studied by decreased capacity to stimulate normal autoreactive T-cel...

1,3-Bis(chloroethyl)-1-nitrosourea (BCNU) has been shown to "cure" over 90% of the mice bearing the syngeneic tumor LSA, and the cured mice acquire elevated levels of tumor-specific immunity. In the present study, we report for the first time the establishment and characterization of several tumor-specific CD8+ cytotoxic T cell (CTL) clones from splenic T cells of BCNU-cured LSA mice. Many of these clones were found to be strongly cytotoxic to LSA but not to a different H-2b tumor target such as EL-4, or the natural killer (NK)-susceptible target YAC-1, NK-resistant target P815, or con A or LPS blasts from H-2b mice. Some of the clones showed a moderate level of cytotoxicity to the NK-susceptible target YAC-1. The relative roles of interleukins such as IL-2, IL-4 or IL-6 in supporting the proliferative response of some LSA-activated CTL clones were analyzed. As expected, recombinant human (rh) IL-2 alone supported the proliferative response of activated CTL clones. Additio...

Cell-mediated immunity (CMI) to homologous spermatozoal antigens was studied in sixty-two males following vasectomy of a duration of 1-10 years. A group of twenty-two normal, fertile non-vasectomized males was also included in the study. The inhibition in the leucocyte migration test (LMT), in the presence of spermatozoal antigen, was taken as an index of CMI. Twenty of the sixty-two vasectomized males (32.2 percent) showed a positive LMT reaction. When the results were analysed with reference to the duration of vasectomy, it was noted that four cases each (22.2 percent), showed a positive LMT reaction in the groups 0-2 years, and 3.5 years. On the other hand twelve cases gave a positive reaction in the group 6-10 years (46.1 percent). It appears that the incidence of CMI to spermatozoa increases with the duration in vasectomy.

Plant-derived cannabinoids, including #9-tetrahydrocannabinol (THC), induce apoptosis in leukemic cells, although the precise mechanism remains unclear. In the current study, we investigated the effect of THC on the upstream and downstream events that modulate the extracellular signal-regulated kinase (ERK) module of mitogen-activated protein kinase pathways primarily in human Jurkat leukemia T cells. The data showed that THC down-regulated Raf-1/mitogen- activated

Summary The lprgeneinducesinmice,accumulationoflargenumbers ofCD4 -CD8 - (doublenegative (DN)) T lymphocyteswhich bearadhesionmoleculesnot characteristic ofnormal restingT cells .Thesecellsfailtoacquireinterleukin 2 (IL-2)receptors, produceIL-2,andproliferate when activated with mitogensormonoclonalantibodies(mAbs) againsttheT cellreceptor(TCR) . Becauseofthesepoorfunctions invitro, thenatureandsignificance ofDN T cells intheautoimmune diseaseprocessisnotclear.Inthecurrentstudy,we describeasurprisingfindingthatmAbs againstCD3-TCR-tx/(3 complex canstronglytriggerthelyticactivity oftheDN T cellsto induceredirected lysisofFcreceptor-positive targets .Similarredirected lysiswas alsoinducible usingmAbs againstCD44 and gp90Mt :L-14,moleculesinvolvedinthebindingoflymphocytes toendothelial cells .The spontaneouscytotoxic potential oftheDN T cells

3,3'-Diindolylmethane (DIM) is a naturally derived indole found in cruciferous vegetables that has great potential as a novel and effective therapeutic agent. In the current study, we investigated the effects of DIM post-treatment on the regulation of activated T cells during the development of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. We demonstrated that the administration of DIM 10 days after EAE induction was effective at ameliorating disease parameters, including inflammation and central nervous system cellular infiltration. MicroRNA (miRNA) microarray analysis revealed an altered miRNA profile in brain infiltrating CD4(+) T cells following DIM post-treatment of EAE mice. Additionally, bioinformatics analysis suggested the involvement of DIM-induced miRNAs in pathways and processes that halt cell cycle progression and promote apoptosis. Additional studies confirmed that DIM impacted these cellular processes in activated T cells. ...

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.

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... Immunotherapy for Glioblastoma Azizul Haque, Mitzi Nagarkatti, Prakash Nagarkatti, Naren L. Banik, and Swapan K. Ray Abstract The most common and deadliest brain tumor is glioblastoma, which escapes immune recognition and kills the patients with a year of diagnosis. ...

Memory T-cell populations in human antitumor tumor-infiltrating lymphocytes (TILs) for adoptive cell transfer have not been fully characterized. Our studies demonstrated that CD62L, CD27 and CD28 positive effector memory T-cells were present in the TIL samples from the tumor tissues of melanoma patients and T-cell expansion led to the significant loss of memory T-cells. CD27- and CD28-positive T-cells had high levels of CD44 expression. T-Cell expansion resulted in significant down-regulation of CD44 expression. Interleukin-2 (IL-2) and anti-CD3 antibody stimulation may be responsible for CD44 down-regulation on CD8(+) T-cells during expansion. Furthermore, CD44 down-regulation using small interfering RNA (siRNA) on TILs dramatically reduced interferon-gamma and IL-2 release upon tumor stimulation. These results suggest that the regulation of CD44 expression in TILs may play an important role in memory T-cell maintenance and antitumor immune response.

Dietary indole derivatives, indole-3-carbinol (I3C) and diindolylmethane (DIM), possess anti-cancer properties and exhibit the characteristics of aryl hydrocarbon receptor (AhR) ligands. Because AhR activation has recently been shown to regulate T cell differentiation, we tested the hypothesis that I3C and DIM may mediate anti-inflammatory properties by promoting regulatory T cell (T-regs) differentiation while inhibiting Th17 cells. We investigated the therapeutic efficacy of I3C and DIM against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The efficacy was evaluated based on clinical scores of paralysis, histopathology, serum cytokines and infiltration of T cells in the CNS. We next studied the mechanism of induction of T cells against myelin oligodendrocyte glycoprotein (MOG₃₅₋₅₅ ) peptide, both in vivo and in vitro, specifically investigating the differentiation of T-regs and Th17 cells, and determined if indoles were acting through AhR. Pretreatment of EAE mice with I3C or DIM completely prevented the clinical symptoms and cellular infiltration into the CNS. Also, post-treatment of EAE with I3C or DIM proved highly effective in curtailing the overall severity of the disease. In addition, I3C or DIM promoted the generation of T-regs, while down-regulating the induction of MOG-specific Th17 cells. The regulation of FoxP3 induction and suppression of Th17 cells by indoles in vivo and in vitro were found to be AhR-dependent. Together, our studies demonstrate for the first time that I3C and DIM may serve as novel therapeutics to suppress neuroinflammation seen during MS through activation of AhR.

The aim of this review is to discuss research involving ligands for the aryl hydrocarbon receptor (AhR) and their role in immunomodulation. While activation of the AhR is well known for its ability to regulate the biochemical and toxic effects of environmental chemicals, more recently an exciting discovery has been made indicating that AhR ligation can also regulate T-cell differentiation, specifically through activation of Foxp3(+) regulatory T cells (Tregs) and downregulation of the proinflammatory Th17 cells. Such findings have opened new avenues of research on the possibility of targeting the AhR to treat inflammatory and autoimmune diseases. Specifically, this review will discuss the current research involving natural and dietary AhR ligands. In addition, evidence indicating the potential use of these ligands in regulating inflammation in various diseases will be highlighted. The importance of the AhR in immunological processes can be illustrated by expression of this receptor on a majority of immune cell types. In addition, AhR signaling pathways have been reported to influence a number of genes responsible for mediating inflammation and other immune responses. As interest in the AhR and its ligands increases, it seems prudent to consolidate current research on the contributions of these ligands to immune regulation during the course of inflammatory diseases.

Autoimmune-susceptible, MRL-lpr/lpr (lpr) mice develop a profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in peripheral lymphoid organs. The source and the mechanism of this abnormal accumulation of cells is still unknown. Recently, we reported that a significant number (approximately 35%) of the CD4-CD8- cells expressed J11d, a marker expressed by immature thymocytes but not by mature functional peripheral T cells. In the present study, we investigated the phenotype, growth requirements, and functional properties of purified J11d+ and J11d- subpopulations. Using the mAb, F23.1, which recognizes a TCR determinant encoded by the V beta 8 gene family, it was observed that approximately 30% of the J11d+ and J11d- DN cells expressed this determinant. Further studies on the thymus revealed that J11d+ DN cells from lpr thymus also contained F23.1+ cells (approximately 25%), whereas, similar cells from normal MRL(-)+/+mice were all F23.1-, consistent with earlier reports in other normal strains. Further phenotypic studies revealed that the peripheral J11d+ and J11d- cells from lpr mice were similar in expressing CD3, Ly-5 (B220), and Ly-24 (Pgp-1) determinants. When stimulated with phorbol myristic acetate (PMA) and recombinant IL-2 (rIL-2), only J11d- cells but not J11d+ cells responded by proliferation. However, in the presence of calcium ionophore (A23187) and PMA, both J11d+ and J11d- subpopulations proliferated by producing and responding to endogenous IL-2 but not IL-4. The lymph node T cells from 1-month-old MRL-lpr/lpr mice responded strongly when stimulated with PMA + rIL-4 or PMA + rIL-6. In contrast both J11d+ and J11d- subpopulations failed to respond when similarly stimulated. The J11d+ but not J11d- cells demonstrated spontaneous cytotoxic activity against the NK-sensitive YAC-1 tumor targets. The J11d- cells did not exhibit cytotoxic potential in spite of culture with PMA + rIL-2. Even after repeated culture in vitro with PMA + A23187 or PMA + rIL-2, both J11d+ and J11d- subpopulations failed to express the mature phenotype bearing CD4 and/or CD8 antigens. The present study demonstrates the expansion of unique J11d+, alpha beta-TCR+, DN T cells with cytotoxic potential in lpr mice and further suggests the existence of phenotypic and functional heterogeneity among the abnormal lpr DN cells.

In the current study, we investigated the effect of growth of FasL(+) tumors in vivo on the functions of peripheral lymphoid organs and the liver. Injection of FasL(+) LSA tumor cells into syngeneic C57BL/6 wild-type mice but not C57BL/6 lpr/lpr (Fas-deficient) mice caused apoptosis in splenocytes. Spleen cells expressing CD3, CD4, CD8, CD19, Mac-3, and CD44 were all susceptible to tumor-induced apoptosis. Also, activated T cells were more sensitive to apoptosis induced by LSA tumor cell lysate when compared to naïve T cells. In contrast, anti-Fas Abs (Jo2) induced apoptosis in only activated but not naïve T cells. When the LSA tumor-bearing mice were injected with a superantigen (SEA), these mice showed a significant decrease in the expansion of SEA-reactive Vbeta3(+) and Vbeta11(+) T cells. When injected into syngeneic mice, the FasL(+) LSA tumor cells caused hepatotoxicity, as indicated by an increase in serum aspartate aminotransferase (AST) levels. Interestingly, Fas-deficient C57BL/6 lpr/lpr mice also showed significant AST levels in the serum following LSA tumor growth. Moreover, hepatocytes isolated from C57BL/6 wild-type and C57BL/6 lpr/lpr mice were equally susceptible to apoptosis induced by LSA tumor cell lysate in vitro. Using cDNA array, LSA tumor cells were found to express several cytokine genes including IL-2, IL-7, IL-11, IL-13, IL-16, lymphotoxin beta, and tumor necrosis factor beta. Together, these data suggested that, in mice bearing FasL(+) LSA tumor, the immunotoxicity is FasL-based, whereas the hepatotoxicity, at least in part, may be FasL-independent.