Normal Splenocytes Research Articles

Suppressive effects of IPD-1151T (suplatast-tosilate) on induction of mast cells from normal mouse splenocytes

The influence of IPD-1151T (suplatast-tosilate) on murine mast cell induction was examined by in vitro cell culture. Splenocytes from BALB/c mice suspended in RPMI-1640 medium supplemented with interleukin-3 were cultured in the presence or absence of IPD-1151T. Half of the total volume of the medium was changed every 4–5 days. The number of mast cells increased with culture time and reached a peak on the 16th day when the cells were cultured without IPD-1151T. However, the growth of mast cells from the splenocyte culture was dose dependently inhibited by IPD-1151T. Futhermore, IPD-1151T inhibited the proliferation of mast cell progenitor cells, IC-2, but not that of splenocytes of mature mast cells.

The role of tumor necrosis factor and interferon gamma in graft-versus-host disease and related immunodeficiency.

The role of TNF in the expression of GVHD and GVHD-related immunodeficiency was studied in a well-established murine GVHD model of bone marrow transplantation across minor histocompatibility barriers (B10.BR-->GBA/J) both in vitro and in vivo. Splenocytes from animals with GVHD profoundly inhibited the proliferation of normal spleen cells in response to a wide range of stimuli in an MHC-nonrestricted fashion. Neutralizing mAbs to TNF reversed the ability of splenocytes from animals with GVHD to suppress the proliferation of normal splenocytes stimulated by the mitogen concanavalin A. Addition of rTNF enhanced the degree of suppression. This reversal was similar to that previously reported for IFN gamma and leucine methyl ester treatment of the GVHD populations. All three components are necessary for suppression to occur because addition of rTNF to cultures in which suppression had been reversed by anti-IFN gamma or leucine methyl ester treatment did not reconstitute suppression. Neutralization of endogenous TNF production in vivo resulted in an amelioration of clinical GVHD, but neutralization of endogenous IFN gamma resulted in a more severe course. However, in vivo neutralization of either TNF or IFN gamma post-BMT resulted in a decreased ability of splenocytes from animals with GVHD to suppress mitogen responses but did not affect the generation of the suppressor cell population. These findings support multiple roles for TNF and IFN gamma in the pathophysiology of GVHD, including terminal cellular differentiation and/or regulation of effector cell function.

Immunosuppression in adult female B6C3F1 mice by chronic exposure to ethanol in a liquid diet

The overall objective of these studies was to characterize the effects of ethanol on the immunocompetence of adult female B6C3F1 mice. To obtain a significant suppression in the antibody response to SRBC, splenocytes from untreated mice had to be directly exposed to concentrations of ethanol from 0.3% to 3.0%, or to acetaldehyde at concentrations greater than 0.03%. We do not believe that these results are consistent with a role by a direct effect by either ethanol or its primary metabolite because these concentrations are higher than what could be obtained as reasonable blood levels. For in vivo exposure, we employed a pair-feeding regimen which was based on a liquid diet containing 5% ethanol (v/v) that provided 36% of the caloric intake as ethanol. Our results indicated that there was a definite temporal relationship to the consequent suppression of the antibody response to SRBC in that no effect was observed after 14 days exposure, and that the magnitude of the suppression increased from 18% after 21 days to 70% after 42 days. We also monitored the liver for histopathology and observed that the ethanol-induced liver damage was restricted to steatosis (fatty liver), which was also manifested with time and which was most pronounced after 42 days exposure. In contrast to our results with the in vivo antibody response, we saw no effect on mitogen-induced proliferation by splenocytes from ethanol-treated mice. These results promped us to measure in vitro antibody responses by splenocutes from ethanol-treated mice. We saw no suppression of the in vitro antibody responses to SRBC, DNP-Ficoll or LPS after any length of exposure to ethanol, and speculated that the basis for the suppression of the in vivo antibody response was an indirect consequence of exposure. We subsequently determined that when normal splenocytes were cultured in 5% serum from ethanol-exposed mice (42-day group), there was a > 80% suppression relative to the serum from the pair-fed controls. As important controls for these studies, we have demonstrated that there was no difference between the responses of normal lymphocytes cultured in 5% normal mouse serum and in 5% serum taken from the pair-fed restricted controls. A determination of the ethanol content in the serum from ethanol-exposed mice (42-day group) indicated that the amount of ethanol present in these cultures was < 0.003%. These results suggest that the mechanism of ethanol-induced immunosuppression is at least in part due to an indirect consequence of chronic exposure, which is possibly mediated by a serum factor.

Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation.

The Tpl-2 locus, cloned by provirus tagging from one of three sublines of the Moloney leukemia virus-induced rat thymoma 2769, defines a gene encoding a protein kinase associated with progression in 22.5% of the tumors. Tpl-2 is expressed primarily in spleen, thymus, liver, and lung. Provirus integration occurs in the last intron of the gene, leading to the expression of a truncated mRNA that terminates in the proviral long terminal repeat and encodes a protein with an altered C-terminal domain. Strong evidence that this genetic change confers growth advantage to affected cell clones was provided by the finding that, during cultivation of all three sublines derived from tumor 2769, cells were selected that harbored independent provirus insertions in the Tpl-2 locus. Exposure of normal rat spleen cells to Con A induces the expression of enhanced levels of Tpl-2 within the first 60 min from the time of exposure suggesting that, in normal splenocytes, Tpl-2 may be involved in the transition from a quiescent to the G1 phase of the cell cycle.

Modulation of macrophage membrane phospholipids by n-3 polyunsaturated fatty acids increases interleukin 1 release and prevents suppression of cellular immunity following hemorrhagic shock.

Studies have suggested that the significant suppression of cellular immunity following hemorrhage may be due to an increased release of prostaglandin E2 (PGE2) by macrophages. Since diets high in n-3 polyunsaturated fatty acids decrease PGE2 synthesis, we assessed whether hemorrhage-induced immunosuppression could be prevented by dietary manipulation. C3H/HeN mice were fed for 3 weeks with fat sources derived from corn oil, safflower oil, or fish oil, then bled to a mean blood pressure of 35 mm Hg maintained for 60 minutes. Following this, the animals were adequately resuscitated with fluids and killed 24 hours later. In the corn oil and safflower oil groups, hemorrhage resulted in a significant increase in PGE2 release by peritoneal macrophages, a marked suppression of peritoneal macrophage antigen presentation capacity, interleukin 1 release, splenocyte proliferation, and interleukin 2 secretion compared with shams. However, feeding mice with fish oil for 3 weeks prior to hemorrhage prevented the rise in PGE2 release and maintained normal macrophage and splenocyte functions following hemorrhage. Thus, the elevated release of PGE2 by peritoneal macrophages plays a pivotal role in hemorrhage-induced immunosuppression. Moreover, diets high in n-3 polyunsaturated fatty acids may offer a new therapeutic approach for preventing posthemorrhage immunosuppression and increased mortality from sepsis.

Induction of tolerance to autoimmune diabetes with islet antigens.

The development of T cell tolerance to self-antigens is imparted principally through negative selection events during thymic ontogeny. However, this tolerance may be limited to antigens that are expressed in the thymus, and additional mechanisms are probably required to regulate autoimmune responses to tissue-specific antigens. Autoimmune diabetes can be induced experimentally by treating susceptible stains of mice with multiple low doses of streptozotocin (STZ). In this report we show that transplantation of isolated islets of Langerhans into the thymuses of adult C57BL/KsJ mice will induce tolerance to the subsequent induction of autoimmune diabetes. This tolerance is tissue specific and thymus dependent. It was not induced by thymic transfer of adrenal tissue or by kidney transfer of islets. Furthermore, depletion of mature T cells was required and the tolerant state was abrogated by the adoptive transfer of normal splenocytes. It is interesting that pretreatment of the islets with STZ enhanced their ability to induce tolerance, and suggests that antigen shedding induced by tissue damage may facilitate transfer of islet antigens to tolerizing cells in the thymus. These findings indicate that thymic tolerance specific for tissue can be stimulated to occur in the presence of atopic tissue-specific intrathymic antigens. Elimination of disease-related T cells in the absence of global immunosuppression represents a novel approach for the prevention of autoimmune disease.

Enhanced glutamine and glucose metabolism in cultured rat splenocytes stimulated by phorbol myristate acetate plus ionomycin

Metabolism of glutamine and glucose was studied in normal rat splenocytes cultured for 48 hours in the presence and absence of a mixture of the mitogens, phorbol myristate acetate (PMA) + ionomycin (Iono). 3H-Thymidine uptake by splenocytes was stimulated more than 500-fold by PMA + Iono. After culture, cells were incubated for 2 hours in the presence of either 2 mmol/L [U- 14C]glutamine ± 5 mmol/L glucose or 5 mmol/L [U- 14C]glucose ± 2 mmol/L glutamine in Krebs-Ringer HEPES buffer. Glutamine was metabolized mainly to ammonia, glutamate, aspartate, and CO 2, and these products were all increased ( P < .01) by twofold to 2.5-fold in stimulated cells. Glucose was metabolized mainly to lactate and to a lesser extent, to pyruvate and CO 2. Lactate production from glucose was increased ( P < .01) by 2.4-fold in stimulated cells, without changes in pyruvate or CO 2 production. In unstimulated, cultured splenocytes, glutamine was not quantitatively as important as glucose in the provision of adenosine triphosphate (ATP), as calculated on the basis of measured metabolites. However, in stimulated cells, glutamine became a much more important energy substrate, providing similar amounts of ATP to those from glucose. The oxidation of glutamine via the Krebs cycle was the major pathway for glutamine-derived ATP production, while lactate production from glucose accounted for the major part of glucose-derived ATP in PMA + Iono-stimulated splenocytes. Thus, we suggest glutamine plays a dual metabolic role in these cells, as both an important fuel and an essential source of carbon and nitrogen precursors for biosynthetic processes. Given the relative mass of cells of the immune system in the body, our findings suggest both the need for an adequate supply of glutamine for the functions of immunocytes in states of altered nutrition and stress, and the significant contribution of glucose to circulating lactate by these cells.

Interaction of fibronectin and fibronectin binding protein (FnBP) of Staphylococcus aureus with murine phagocytes and lymphocytes.

In the present study we examined the in vitro and in vivo interactions of a cloned staphylococcal fibronectin binding protein (FnBP) and plasma fibronectin (Fn) with polymorphonuclear cells (PMNs) and macrophages, and how antibodies against FnBP affect the phagocytosis process in vitro. Moreover, the interaction of FnBP and Fn coupled on latex beads as 'artificial bacteria' and 'artificially opsonized bacteria' with murine spleen cells and peritoneal macrophages was tested. The major finding of the present study is that antibodies against the FnBP of Staphylococcus aureus (S. aureus) and low concentration of antibodies recognized two IgG-binding domains of protein A (SpA) are effective in the promotion of phagocytosis in vitro. It was also observed that FnBP has a chemoattractant activity and causes accumulation of PMNs and macrophages in the mouse peritoneal cavity when injected 24 h before irritation of peritoneal exudate. It seems likely that this activity is connected with binding to Fn molecules since formalin inactivation of FnBP (60%) abolished it. In the in vitro phagocytosis assay in the presence of FnBP (in a medium supplemented with serum depleted of Fn), ingestion of bacteria by phagocytes was identical to assay carried out in the presence of BSA. However, addition of plasma fibronectin caused an increased uptake of bacteria by macrophages and to a lesser degree by PMNs. We observed that in a population of normal splenocytes, those cells that effectively bound FnBP- and Fn-coated latex beads were mostly those cells exhibiting macrophage and dendritic morphology. In populations of spleen cells of animal infected with S. aureus, T lymphocytes were also found to bind FnBP- and Fn-coated latex beads. These data suggest that FnBP may have the ability to promote aggregation of immune cells, either directly or by interaction with plasma Fn, which can be helpful in certain cell-cell interactions taking place at the initial stages of specific immune response.

Interaction of fibronectin and fibronectin binding protein (FnBP) of Staphylococcus aureus with murine phagocytes and lymphocytes

In the present study we examined the in vitro and in vivo interactions of a cloned staphylococcal fibronectin binding protein (FnBP) and plasma fibronectin (Fn) with polymorphonuclear cells (PMNs) and macrophages, and how antibodies against FnBP affect the phagocytosis process in vitro. Moreover, the interaction of FnBP and Fn coupled on latex beads as ‘artificial bacteria’ and ‘artificially opsonized bacteria’ with murine spleen cells and peritoneal macrophages was tested. The major finding of the present study is that antibodies against the FnBP of Staphylococcus aureus (S. aureus) and low concentration of antibodies recognized two IgG-binding domains of protein A (SpA) are effective in the promotion of phagocytosis in vitro. It was also observed that FnBP has a chemoattractant activity and causes accumulation of PMNs and macrophages in the mouse peritoneal cavity when injected 24 h before irritation of peritoneal exudate. It seems likely that this activity is connected with binding to Fn molecules since formalin inactivation of FnBP (60%) abolished it. In the in vitro phagocytosis assay in the presence of FnBP (in a medium supplemented with serum depleted of Fn), ingestion of bacteria by phagocytes was identical to assay carried out in the presence of BSA. However, addition of plasma fibronectin caused an increased uptake of bacteria by macrophages and to a lesser degree by PMNs. We observed that in a population of normal splenocytes, those cells that effectively bound FnBP- and Fn-coated latex beads were mostly those cells exbiting macrophage and dendritic morphology. In populations of spleen cells of animals infected with S. aureus, T lymphocytes were also found to bind FnBP- and Fn-coated latex beads. These data suggest that FnBP may have the ability to promote aggregation of immune cells, either directly or by interaction with plasma Fn, which can be helpful in certain cell-cell interactions taking place at the initial stages of specific immune response.

Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts

This study was undertaken to characterize the phenotype and function of lymphocytes derived from endomyocardial biopsies in heart transplant patients. To this aim, tissue infiltrating lymphocytes were derived from seven heart transplant patients and were analyzed for the expression of a panel of markers, including CD3, CD4, CD8, CD16, CD56, CD45RA, CD45RO, α β and γ δ T cell receptor, and for their ability to lyse a series of targets, including NK-sensitive K-562 targets, NK-resistant Raji targets, donor related, and unrelated normal splenocytes. Our data show that the majority of cultured lymphocytes expressed the CD3 + phenotype and the α β T cell receptor. The CD4 and CD8 molecules were heterogeneously expressed among T cell lines tested. Concerning cytotoxic related markers, a significant percentage of cells were CD56 +. The evaluation of CD45 isoforms showed that both “naive” and “memory” cells were present among heart TIL. Cytotoxic in vitro studies demonstrated that all our T cell lines showed an efficient cytotoxic machinery when tested against NK-sensitive targets. A marked lysis of donor-related splenocytes was demonstrated in all patients tested. To investigate the role of CD3 and HLA class I molecules in the cytotoxic mechanisms taking place in human heart allograft rejection mechanisms, TIL were assessed for their lytic activity against different targets in the presence of anti-CD3 and anti-HLA class I monoclonal antibodies (mAbs). Although donor-specific cytotoxicity was considerably inhibited by the anti-CD3 mAb, no inhibitory effect was displayed by this antibody on TIL-mediated cytotoxicity against donor-unrelated splenocytes. Anti-HLA class I mAb was able to inhibit both allospecific and nonallospecific cytotoxicity. These data suggest that different types of cytotoxic cells may be propagated from biopsy specimens of heart transplant patients.

Altered prolactin response of the lymphocytes of tumor‐bearing mice

Interaction of prolactin and glucocorticoid on thymocytes and splenocytes of normal and tumor-bearing mice with reference to their mitogen-responsive blastogenesis has been studied. Prolactin alone had little or no mitogenic effect on thymocytes and splenocytes of normal mice but it was co-stimulatory, with ConA, in inducing blastogenesis in normal splenocytes. Thymocytes and splenocytes of tumor-bearing mice responded differently to prolactin. Hormone alone inhibited growth of thymocytes but at certain concentrations stimulated splenocytes. When cultured with prolactin and ConA, the thymocytes of tumor hosts responded with increased proliferation compared to that induced by ConA alone. Glucocorticoid suppressed ConA-induced lymphocyte proliferation in both normal and tumor-bearing mice. Prolactin reversed the inhibitory effect of glucocorticoid in normal mice but failed to abrogate inhibition in tumor hosts. Altered responsiveness of lymphocytes of tumor hosts to prolactin was not a function of circulating prolactin, as the serum prolactin level was similar in normal and tumor-bearing mice. Prolactin, however, could not reverse estradiol-induced suppression of lymphocyte proliferation. The lactogenic hormone, but not somatogenic hormones, altered the glucocorticoid inhibition of lymphocyte growth.

Monoclonal antibodies to feline lymphocyte membranes recognize the leukocyte-common antigen (CD45R).

By immunization of BALB/c mice with a feline T lymphoblastoid cell line, MYA-1 cells, two types of lymphocyte-specific monoclonal antibodies (mAbs) were obtained. The 220/205/190 kd protein defined by 2F11 mAb is highly expressed on the surface of MYA-1 cells and another feline T lymphoma cell line, FL74 cells. The protein is also expressed on normal feline thymocytes, splenocytes and feline peripheral blood mononuclear cells (PBMCs). Another mAb, 17B10, caused similar results as those of 2F11 except for its low reactivity with FL74 cells. The second type of mAb, 15B3, defined the 220 kd protein. The reactivities of this mAb with MYA-1 cells, FL74 cells, PBMCs and feline splenocytes were lower than the former two mAbs, and did not react to feline thymocytes. On the other hand, 17B10 and 15B3 defined partial populations of MYA-1 and FL74 cells recognized by 2F11. The cells defined by the 2F11 and 17B10 are all leukocytes in spleen and lymph node. In contrast, 15B3 defined most of the cells in B cell area and partially in T cell area. These results suggested that 2F11 and 17B10 recognized the specific antigen of 220/205/190 kd of the leukocyte-common antigen (L-CA) family, CD45R, with different epitopes, and that 15B3 defined the distinct antigen of 220 kd on CD45R.

Suppressor and protector factors derived from Trypanosoma lewisi

Ndarathi C. M. 1991. Suppressor and protector factors derived from Trypanosoma lewisi. International Journal for Parasitology 21: 763–769. Trypanosoma lewisi is a specific protozoan blood parasite of rats. Normal rats infected 2 h after treatment with plasma from day 8 irradiated (8.5 Gy) infected rats had significantly higher parasitaemia; in contrast, animals infected 7 days post-plasma treatment were significantly protected. Trypanolytic and ablastic antibodies could be demonstrated in the serum of normal rats treated with the plasma; the trypanolytic antibodies were stage-specific. Suppression of normal rat splenocyte responses to Con A and lipopolysaccharide (LPS) stimulation were also observed in the presence of different protein concentrations of whole lysate from epimastigote forms. The suppression by mitogen Con A was ablated by the addition of exogenous IL 2, or by washing cells incubated with the lysate prior to mitogen stimulation. These results indicate that immunoregulatory factors are present in the plasma of rats infected with T. lewisi, and the effect of these factors can be demonstrated in vitro with whole parasite lysate. The restoration of normal splenocyte responses to Con A by addition of exogenous IL 2 or by washing cells suggests that the suppressor factor(s) act(s) on the T cells by inhibiting their proliferation and IL 2 production, and the continued presence of these products is essential in the maintenance of suppression.

A role for gamma interferon, tumor necrosis factors, and soluble T-cell receptors in the depressed blastogenic response of spleen cells of Mycobacterium lepraemurium-infected mice

Spleen cells of Mycobacterium lepraemurium-infected mice were cultured on petri dishes coated with mycobacterial antigens, and antigen-reactive cells were isolated. Upon incubation in mitogen- or antigen-free culture medium, these cells released mediators capable of depressing the in vitro proliferative response of normal splenocytes to specific antigen and to concanavalin A and lipopolysaccharide. One of these mediators was identified with gamma interferon (IFN-gamma), mainly on the basis that treatment of supernatants with monoclonal anti-IFN-gamma antibodies markedly reduced the suppressive activity contained therein. Detectable levels of tumor necrosis factor alpha (TNF-alpha) and TNF-beta were present in spleen cell culture supernatants of infected mice. Moreover, low doses of recombinant TNF-alpha and TNF-beta were found to potentiate the suppressive activity of exogenous IFN-gamma. Soluble T-cell receptors beta were also detected in the culture supernatants. The elimination of these molecules with monoclonal anti-T-cell receptor beta (F23.1) antibodies immobilized on a plastic surface partially reversed the depression of the response to mycobacterial antigen but did not affect the response to mitogens. These results revealed the complex nature of suppressor mediators that are produced by mycobacterial antigen-reactive cells and that regulate the in vitro proliferative response.

Immunosuppression induced by attenuated Salmonella. Reversal by IL-4.

We previously demonstrated that an aroA- strain of Salmonella typhimurium, which provides excellent protection against virulent Salmonella challenge, also rendered immunized mice unable to mount in vivo and in vitro antibody responses to heterologous Ag. Coculture studies using transwell plates indicated that suppression was mediated by soluble factors. The suppressive cells were identified as belonging to the monocytic linkage. Macrophage precursors as well as mature adherent macrophages mediated the observed suppression. In the present study, the mechanism of immunosuppression was investigated. Suppression was found to be genetically nonrestricted as spleen cells from immunized C3HeB/FeJ mice (H-2k) suppressed the anti-SRBC plaque-forming cell (PFC) responses of normal spleen cells from two MHC noncompatible mouse strains, BALB/c (H-2d) and C57BL/6 (H-2b). Time course experiments demonstrated that the addition of spleen cells from immunized mice to normal splenocytes as late as day 4 of a 5-day assay was still markedly suppressive. Furthermore, suppression of the PFC responses was accompanied by a profound inhibition of the capacity of immune splenocytes to produce IL-2 in response to in vitro stimulation by Con A. Coculture studies showed that immune spleen cells were able to suppress IL-2 production by normal splenocytes in a dose-dependent fashion. However, the suppressed PFC responses of immune spleen cells could not be reversed by the exogenous addition of up to 200 U/ml of IL-2, suggesting that immune splenocytes are also defective in their ability to respond to IL-2. In marked contrast, suppression of PFC responses was reduced by more than 50% by the addition of as little as 1 U/ml of IL-4 and was completely abrogated when 5 U/ml of IL-4 were added to in vitro cultures of spleen cells from immunized mice. The antisuppressive action of IL-4 appeared to be via its inhibitory effect on activated macrophages. The implications of the above findings are discussed.

Adoptive Immunotherapy Using Lymphokine-Activated Killer Cells and Recombinant Interleukin-2 in Preventing and Treating Spontaneous Pulmonary Metastases of Syngeneic Dunning Rat Prostate Tumor

Lymphoklne-activated killer (LAK) cells were generated from splenocytes of rats bearing a weakly immunogenic Dunning prostate tumor (R-3327 AT-3) and activated with recombinant interleukin-2 (rIL-2). The maximal LAK activity was obtained from splenocytes of rats bearing tumors for 10 to 14 days after incubation with 1000 U/ml./day of rIL-2 for five to eight days. The majority of these LAK cells expressed high levels of asialo GM1 (89%), laminin (83%), OX-19 (80%) and OX-8 (88%) surface markers. LAK cells exhibited higher cytotoxicity to rat prostate tumor cells and mouse lymphoma In vitro than to other non-prostate tumor cells or normal rat splenocytes and thymocytes. Splenocytes of rats bearing prostate tumors have higher LAK activity than normal splenocytes. The Winn type assay showed that Dunning prostate tumor growth was inhibited effectively by LAK cells at a tumor cell:LAK cell ratio of 1:50. The therapeutic efficacy of LAK cells in the treatment of primary solid prostate tumors and pulmonary metastases of Dunning rats was evaluated. LAK cells in combination with rIL-2 showed a greater therapeutic benefit in 1) prevention of prostate tumor metastases to lung, 2) retardation of the primary tumor growth, 3) regression of spontaneously established pulmonary metastases, and 4) prolongation of survival as compared to untreated controls or those groups treated with LAK cells or rIL-2 alone. The results of this study indicate that the conjunctive therapeutic approach of using surgical therapy to remove primary solid tumors followed by adoptive immunotherapy with LAK cells plus in vivo administration of IL-2 may be potentially valuable in the treatment of prostate tumors, particularly for the spontaneous pulmonary metastases.

Immunomodulatory effect of cimetidine on the proliferative responses of splenocytes from T. cruzi-infected rats

The immunomodulatory effect of cimetidine (CIM), a histamine type-2 receptor antagonist, was evaluated in respect to the blastogenic response to Con A of Wistar Furth (WF) rats infected by the Y strain of Trypanosoma cruzi (T. cruzi). Enhancement of blastogenesis of normal splenocytes was observed at a concentration of 10(3) M. However, the splenocytes from infected animals responded to concentrations of CIM ranging from 10(-8) to 10(-3) M. The mitogenic response to Con A of cells from infected animals was restored in the presence of CIM. The results show that CIM modulates the "in vitro" proliferative response of cells from T. cruzi-infected rats and suggest an immunoregulatory role of histamine and/or of cells that express H2 receptors in this infection.

Lymphokine-activated killer (LAK) cells: VI. NK1.1 +, CD3 + LAK effectors are derived from CD4 −, CD8 −, NK1.1 − precursors

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1 +, CD3 + lytically active subset. These lymphocytes were not derived from NK1.1 + precursors since NK1.1 + cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NKl.1 +, CD3 + effectors were contained in a B cell-depleted CD4 −, CD8 −, NK1.1 − splenic subset. Freshly obtained CD4 −, CD8 −, NK1.1 − splenocytes were mostly CD3 +, CD5 +, B220 −, had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4 −, CD8 −, NK1.1 − splenocytes with IL2, for 6 days, resulted in the development of NK1.1 +, CD3 +, B220 + effectors 40% of which were CD5 dim and 20–25% of which expressed TCR-β8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1 + precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1 + cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1 + lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate tjiat splenic NK cells do not express or acquire CD3; that the NK1.1 +, CD3 + LAK effectors are derived from an NK1.1 − precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.

N-Acetyl-β-endorphin 1–31 antagonizes the suppressive effect of β-endorphin 1–31 on murine splenocyte proliferation via A naloxone-resistant receptor

High affinity binding sites for β-endorphin 1–31 (β-EP) have been observed on transformed mononuclear cells such as the human U937 monocyte-like cell line and the murine EL4-thymoma line, and on normal murine splenocytes. Binding of β-EP at these sites is resistant to competition by naloxone and other opiate receptor ligands but sensitive to N-acetyl-β-endorphin 1–31 (N-Ac), cations and GTP-γ-sulfate. Thus, the following studies were done to determine the functional significance of binding β-EP and N-Ac. β-EP suppressed phytohemagglutinin (PHA)-stimulated [ 3H]thymidine uptake in a dose-dependent, naloxoneinsensitive fashion. β-Endorphin 1–27, (des)-tyrosine β-endorphin 2–31, or N-Ac failed to duplicate the suppressive effect of β-EP. However, N-Ac, which is equipotent to β-EP at displacing 125I-β-EP bound to murine splenocytes or U937 cells, antagonized the suppressive effect of β-EP. Taken together with previous binding studies, the present observations suggest that β-EP effects receptormediated responses on normal immunocytes that do not depend on the activation of neuronal-like opiate receptors which are naloxone-sensitive. N-Ac, which shows minimal binding to such brain opiate receptors, is a potent functional antagonist of the naloxone-insensitive immunocyte receptor for β-EP.

Depression of cell‐mediated immunity in plasmacytoma‐bearing mice

Previous reports have documented that mice bearing plasmacytomas (PC) are suppressed in their ability to mount a primary antibody response, whereas cellular immunity appears to be normal. Studies presented here provide evidence that T cell responsiveness is also depressed in BALB/c mice bearing the Lieberman plasmacytoma (Lpc-1). For instance, splenocytes from mice bearing large tumours were impaired in their in vitro ability to respond to the T cell mitogen, mount an appropriate alloreactive cytolytic T lymphocyte response, and produce interleukin-2 (IL-2). A population of suppressor cells was detected in the spleens 7 days after tumour implantation as evidenced by their ability to prevent normal splenocytes not only from responding to antigens in mixed lymphocyte culture, but also from producing IL-2. A similar inhibitory effect was observed with culture supernatants of these cells, indicating the existence of a soluble suppressive factor. Therefore, the present study demonstrates that cellular immune responses are impaired in mice bearing Lpc-1 tumours and that this effect may be due to the generation of suppressor cells and/or a suppressive factor.