Mouse Splenic Cells Research Articles

Monoclonal antibodies against mouse splenic stromal cells.

A panel of rat monoclonal antibodies directed against mouse splenic stromal cells were isolated. These monoclonal antibodies were immunohistochemically divided into four groups which reacted with non-lymphoid cells of the murine spleen; (I) in the white pulp, (II) at the marginal zone, (III) in the red pulp, and (IV) on the endothelium of splenic blood vessels. These monoclonal antibodies were studied immunohistochemically in lymphoid organs by means of light and electron microscopy. Monoclonal antibodies SS-4 (group I) reacted with fibroblastic reticulum cells that were distributed only in the white pulp of the spleen and in the follicular areas of lymph nodes. The SS-4 staining cell, in clustered splenic stromal cells, formed colonies which included a small number of Thy-1 positive lymphocytes. Therefore, we concluded that SS-4 staining stromal cells comprise the lymphoid compartment. In contrast, monoclonal antibodies SS-1, SS-3 and SS-5 (group II) reacted with dendritic shaped cells in the marginal zone of the spleen. Examination of splenic extramedullary hematopoiesis in mice rescued by bone marrow transplantation after lethal irradiation revealed that SS-3 and SS-5 reacted with dendritic shaped stromal cells in clonal nodules of engrafted marrow in the red pulp. SS-3 and SS-5 staining cells could not be observed in physiologic hematopoiesis of non-transplanted mice. It was suggested that SS-3 and SS-5 staining stromal cells are involved in primitive hematopoiesis. Monoclonal antibodies SS-2, SS-6 and SS-7 (group III) mainly reacted with dendritic cells and macrophages in the red pulp. Monoclonal antibodies SS-B and SS-9 (group IV) reacted with endothelial cells of blood vessels and sinuses. These findings of heterogeneity in mouse splenic stromal cells are further evidence that specific micro-environments are composed by specialized stromal cells.

Trichinella spiralis: Ultrastructural localization of a target antigen recognized by a monoclonal antibody : International Journal for Parasitology Volume 26 (5) (1996), Page 553-555

A cDNA library was constructed from an Angiostrongylus cantonensis young adult and the encoded proteins were expressed in Escherichia coli. One reactive antigen, a RAB-2 protein, was selected using an immunoscreening technique. The expression of the Th1-type cytokine IFN-γ was elicited in mouse splenic cells that were co-cultured with the recombinant RAB-2 protein and in the sera of mice that were immunised with this protein and adjuvant (50 μg at 2-week intervals). In the A. cantonensis-infected groups, the mice were orally infected with 35 infective larvae, and a subset of the infected mice were immunised with the recombinant RAB-2 protein in adjuvant. Serum samples were collected every week for ELISA, and the pathological examinations were performed at 14 days post infection (dpi). An increase in IFN-γ expression was noted in the blood, and the brain sections revealed moderate eosinophilic meningitis in the immunised mice. The RAB-2 antigen of A. cantonensis induced a Th1-type immune response both in vitro and in vivo.

Immunopotentiating activities of polysaccharide fraction from pine seed shell extract.

The function of polysaccharide (PSE) extracted from pine seed shells as a immunopotentiator was investigated. The phagocytosis and chemotaxis of mouse peritoneal macrophages (MP) were augmented by oral administration of PSE. The incorporation of 3H-thymidine by Con A-stimulated mouse splenic cells was significantly intensified by administration of PSE but the adherent-cells (MP)-eliminated splenic lymphocytes was not increased. It is shown that the existence of activated MP is needed for the activation (proliferation) of T lymphocytes. The responsiveness of mouse T cells to alloantigen was also augmented by administration of PSE. The number of anti-SRBC plaque-forming cells in the spleen of mouse and chicken was also significantly increased. The results indicate that MP are first activated by oral administration of PSE and that the activation of T cells is then initiated indirectly by humoral factors (cytokines) produced by activated MP.

Volatile fatty acids, metabolic by-products of periodontopathic bacteria, inhibit lymphocyte proliferation and cytokine production.

Short-chain fatty acids are a major by-product of anaerobic metabolism and can be detected in gingival fluid from periodontal pockets. Since most T cells are present subjacent to the pocket epithelium in conjunction with the plasma cells, it is important to know how these T cells are affected by short-chain fatty acids produced by subgingival plaque. The purpose of this study is to examine the effects of extracellular metabolites from periodontopathic bacteria on the proliferation and cytokine production of mouse splenic cells as a potential mechanism of imbalance among host-microbial interactions. A low-molecular-weight, heat-stable agent present in the two-day culture filtrate of Porphyromonas gingivalis, Prevotella loescheii, and Fusobacterium nucleatum significantly depressed Con A- and LPS- induced cell proliferation. To determine whether short-chain fatty acids present in the filtrate could account for the depression, we tested extracted volatile and non-volatile fatty acids for their effects on mitogenic activity. The volatile fatty acids extracted from immunosuppressive supernatants greatly inhibited T- and B- cell proliferation. Among these volatile fatty acids, butyric, propionic, valeric, and isovaleric acids impaired cell proliferation dose-dependently. From gas-liquid chromatographic analysis data, it is suggested that immuno-inhibitory activities in culture filtrates are mainly attributable to butyric and isovaleric acids in P. gingivalis, to propionic, butyric, and isovaleric acids in P. loescheii, and to butyric acid in F. nucleatum. Furthermore, these fatty acids significantly depressed interleukin 2 (IL-2), IL-4, IL-5, IL-6, and IL-10 production by Con A-stimulated splenic-T cells dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)

A new monoclonal antibody (IE8) reactive with dendritically shaped cells in the human tonsil.

A new monoclonal antibody (mAb), 1E8 (IgG1, kappa), was obtained from a hybridoma prepared by fusion of mouse myeloma cells (NS-1) with splenic cells of mice immunized with a human B blastic malignant lymphoma cell line, HPE-Ret-3 (Ret-3). The mAb showed a reactivity unrestricted to a specific cell lineage on flow cytometrical analysis of the reactivity with human lympho-hematopoietic cell lines. In peripheral blood, 1E8 reacted with the cells of all lineage, that is, lymphocytes, monocytes, granulocytes and platelets, even though its intensity was very low by immunohistochemistry. Immunohistochemical examination of human tonsil with 1E8 showed a characteristic staining pattern. Positive cells scattered in follicular (mantle zone and germinal center), parafollicular (T-dependent area), subepithelial and interstitial connective tissue areas. These positive cells seemed to be categorized into dendritically shaped cells (DSC), including dendritic cells (DC) and a subpopulation of macrophages in follicles, interdigitating cells (IDC) and irregularly shaped mononuclear cells. The localization of 1E8 antigen staining was similar to that of integrin CD11c, although its distribution on hematopoietic cell lines did not coincide with that of 1E8 antigen. Immunobiochemical studies showed that 1E8 bound two cell surface proteins with molecular size of 70,000-90,000 and 35,000 Da each. Consequently, 1E8 antigen might be a novel marker of DSC.

IL-10 inhibits the primary allogeneic T cell response to human peripheral blood dendritic cells.

Most of the immunosuppressive properties of IL-10 are related to its action on antigen presenting cells (APC). Until now, several mechanisms have been described by which IL-10 inhibits the ability of monocytes/macrophages to induce T cell activation. Indeed, IL-10 down-regulates their expression of MHC class II molecules1 as well as of ICAM-1 and B7-1 accessory molecules2,3. Moreover, the inhibition by IL-10 of IL-12 and IL-1 synthesis by monocytes was shown to be responsible for the inhibition of IFN-y production by peripheral blood mononuclear cells (PBMC)4. As far as the effects of IL-10 on dendritic cells are concerned, IL-10 was shown to inhibit the ability of mouse splenic dendritic cells to induce interferon (IFN)-y production by antigen-specific TH1 clones or alloreactive splenic T cells5. In addition, human epidermal Langerhans cells preincubated with IL-10 were found to be deficient in the triggering of a mixed lymphocyte reaction6. As a first approach to study the effects of IL-10 on human peripheral blood dendritic cells (PBDC), we analyzed the influence of IL-10 on a primary allogeneic T cell response induced by those cells.

Very late activation antigen 4-vascular cell adhesion molecule 1 interaction is involved in the formation of erythroblastic islands.

Erythroblastic islands are anatomical units consisting of a central macrophage surrounded by erythroblasts. We studied the adhesion molecules involved in the formation of these structures. Central macrophages of erythroblastic islands isolated from the spleens of phlebotomized mice were clearly stained for vascular cell adhesion molecule 1 (VCAM-1). The surrounding erythroblasts of the erythroblastic islands strongly expressed the alpha 4 integrin of very late activation antigen 4 (VLA-4: alpha 4 beta 1 integrin), the counter receptor of VCAM-1, whereas most reticulocytes and erythrocytes did not. Both monoclonal antibodies (mAbs) against alpha 4 integrin and VCAM-1 disrupted the erythroblastic islands cultured in the presence of erythropoietin. Moreover, adhesion of splenic erythroblasts to tumor necrosis factor alpha-stimulated mouse splenic endothelial cells, which showed high expression of VCAM-1 but not intercellular adhesion molecule 1, was inhibited by the anti-VCAM-1 and anti-alpha 4 mAbs. These findings suggest that VLA-4-VCAM-1 interaction plays a crucial role in the formation of erythroblastic islands.

Triptolide suppresses T-lymphocyte proliferation by inhibiting interleukin-2 receptor expression, but spares interleukin-2 production and mRNA expression

The purpose of this study was to elucidate the mechanism of action of triptolide on the T-lymphocyte-mediated immune response. Lymphocytes were incubated with a suboptimal dose oof Con A or PHA in the presence or absence of varying doses of triptolide to assess the effect of triptolide on lymphocyte proliferation, interleukin-2(IL-2) production and IL-2 receptor expression. Then, Con A or PHA induced T-blast cells were cultured with a sufficient dose of recombinant human IL-2 in the presence or absence of triptolide to evaluate the effect of triptolide on the interaction of IL-2 and IL-2 receptors. The effect of triptolide on the immune response in vivo was also investigated. The results of these studies clearly demonstrated that triptolide selectively inhibited the T-lymphocyte proliferative response to Con A and PHA, but had less effect on LPS-induced B-lymphocyte proliferation. Triptolide also suppressed the expression of IL-2 receptors on PHA induced T-blast cells, but did not alter the production of IL-2 by mouse splenic cells and human tonsil lymphocytes. Furthermore, the results also showed that triptolide at higher concentration had a slight inhibitory effect on the interaction of IL-2 and IL-2 receptors, and addition of exogenous IL-2 did not reverse the inhibiting action of triptolide on T-cell proliferation. Taken together, these results suggest that triptolide inhibits T-lymphocyte proliferation mainly by affecting IL-2 receptor expression rather than IL-2 production.

Productions of interferon-γ and nitrite are induced in mouse splenic cells by a heteroglycan-protein fraction from culture medium of Lentinus edodes mycelia

A xylose-rich heteroglycan-protein fraction (LAP1) was prepared from a solid culture medium in which Lentinus edodes mycelia were growing actively. Mouse splenic cells (SPs) were incubated with [3H]TdR in the presence of LAP1. The incubated SPs were fractionated into plastic adherent splenic cells (ADs), nylon-column effluent splenic cells (NEs) and sIg-expressed splenic cells (SIs), which are rich in Mac-1+, Thy-1.2+ and Ly-5+ cells, respectively. The incorporation of [3H]TdR in response to LAP1 was enhanced in each of the fractionated cell populations. Northern or dot blot hybridization showed that productions of IFN-γ and its receptor mRNAs are induced predominantly in NEs. In another experiment, SPs were fractionated into ADs, NEs and SIs. Then, NE-AD, SI-AD and NE-SI mixtures were prepared and incubated in the same manner. A significant incorporation of [3H]TdR was shown only in the NE-AD mixture. The enzyme-linked immunosorbent assay showed that IFN-γ production in response to LAP1 is induced in SPs or in the NE-AD mixture, but not in NEs alone. The level of the production was about 5 times higher in the mixture than in SPs after a 72 h incubation. Moreover, LAP1 was capable of inducing NO2− production in SPs. Thus, the present studies imply that this heteroglycan-protein fraction stimulates productions of IFN-γ and nitrite in mouse splenic cells, augmenting antitumor immune response(s).

A new splice variant of the inositol-1,4,5-triphosphate (IP 3) receptor

In this study we have identified a new splice variant of the IP 3 receptor (IP 3R) transcript in a number of mouse cell lines (e.g. moust T.-lynphoma cells, mouse splenic lymphocytes and mouse NIH 3T3 fibroblast cell lines) using the reverse transcriptase-polymerase chain reacton. This varinet IP 3 receptor (designated as IP 3RV-S2, ≈ 453bp) is larger than the non-neuronal form (402 bp) but smaller than the neuronal form (522 bp) of the IP 3 receptors. Nucleotide sequencing data indicate that this new isoform (IP 3RV-S2) containes a 51 nucleotide insertion within the non-neuronal form of IP 3R at the S2 splice site. During mitogenic stimulation by Con A, the ratio between IP 3R (non-neuronal form) and IP3RV-SE (variant isoform) in mosue splenic T-lymphocytes increased approximately 1.5-fold. The change in relative amounts of these IP 3 receptors isolforms during mitogenic-stimulation suggests that T-lymphocytes may have different requirements for the IP 3 isoforms in order to control intracellular calcium mobilization. The selective expression of these two IP 3R isoforms (IP3RV-S2 and non-neuronal IP 3R) may be critically important for the onset of signal transduction and cell activation.

The costimulating effect of chorionic gonadotropin on lymphokine-activated splenocytes. New aspects of the immunomodulating effect of the pregnancy hormone

An immunodepressive effect of chorionic gonadotropin (CG) low (40IU) and high (200IU) doses on the processes of lymphocyte antigen-independent differentiation, realizing the primary immune response has been established in the short-term culture of mice splenic intact cells. Splenocyte activation by Con-A or human recombinant IL-2 does not show an expressive immunostimulating effect on the processes of plaque forming cells (PFC) formation. While the immunomodulating effect of CG on the background of these activators changes. In the presence of Con-A or IL-2, hormone in the dose of 40 IU can't effectively suppress the PFC formation processes. On the background of IL-2, CG-200IU shows an expressed opposite effect, increasing the splenocyte ability to form PFC twice as much. It is suggested, that CG appears to be a cofactor, intensifying the processes of immunocompetent cell proliferation and differentiation, connected with this lymphokine. A new aspect of the CG immunomodulating effect during pregnancy is being discussed now.

Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4 + helper T cell subsets and allogeneic CD8 + T cells

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th 1 or Th 2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th 1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th 1 helper cells in situ. The remaining six cell lines responded to INF-γ by up-regulating their class II expression and acquiring Th 1 antigenpresenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th 1-derived INF-γ. Five of the cell lines provided accessory cell function to Th 2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th 1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th 1 and Th 2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8 + T cells occurred in a CD4 + helper cell-independent manner and was unaffected by the addition of INF-γ. The alloantigenpresenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.

Mitogenic activities of heteroglycan and heteroglycan—protein fractions from culture medium of Lentinus edodes mycelia

The water soluble material (LEM) was prepared from the solid culture medium in which Lentinus edodes mycelia were growing actively. An alcohol insoluble material was prepared from LEM and subjected to Sepharose 6B gel filtration. The void fraction (LAP1) was composed mainly of xylose-rich heteroglycan and protein. From LAP1, a heteroglycan fraction (LAF1) was prepared by DEAE-Sepharose CL-6B column chromatography. LAP1 and LAF1 enhanced the incorporation of [3H]thymidine into mouse splenic cells (SPs). At each of the optimum doses, the rate of the incorporation was about 10 times as high with LAF1 as with LAP1. Such mitogenic responses were not induced in nylon-column effluent SPs and thymocytes. sIg-expressed cells were responsive to LAF1, but not to LAP1. Moreover, with each fraction, the incorporation was enhaned more in plastic adherent splenic cells (ADs) than in SPs. The flow cytometric assay revealed that the number of Mac-1+ cells is about 13 times as many in ADs as in SPs and that the number of Ly-5+ or Thy-1.2+ cells is considerably reduced in ADs compared with that in SPs. Thus, the present studies suggest that LAP1 and LAF1 act as mitogens predominantly for mouse splenic macrophages and/or monocytes.

Mouse macrophages contain a truncated CD4 transcript.

Mouse macrophages do not express CD4 on their surfaces. We used the polymerase chain reaction to investigate CD4 gene transcription in individual clones of primary mouse splenic macrophages and cell lines of spleen and bone marrow macrophages. The results show only the presence of CD4 mRNA transcripts that are truncated in the 3' coding sequence, thus explaining the lack of expression of a mature CD4 gene product by these cells.

Immunotoxic potential of antiviral drugs: Effects of ganciclovir and ( S)-1-(3-hydroxy-2-phosphonylmethoxy propyl) cytosine on lymphocyte transformation and delayed-type hypersensitivity responses

In the present studies, we examined the in vitro and in vivo effects of ganciclovir (DHPG) and a relatively new nucleoside analogue, ( S)-1-(3-hydroxy-2-phosphonylmethoxy propyl) cytosine (HPMPC), on lymphocyte responses to T cell mitogens and delayed-type hypersensitivity (DTH) responses to dinitrofluorobenzene (DNFB). Initially, responses of mouse splenic mononuclear cells and human peripheral blood mononuclear cells to PHA and con A were evaluated in vitro in the presence of each drug. Both drugs inhibited the responses to each mitogen; however, DHPG had a greater inhibitory effect on con A responses of human and mouse lymphocytes than did HPMPC. Also, spleen cells from mice treated for 7 days with DHPG responded less well to PHA stimulation than cells from untreated or HPMPC-treated mice. No effect of either drug was observed on con A responses. Treatment of mice with either drug decreased the development of DTH responses, with HPMPC having a greater inhibitory effect than DHPG. The results from the present studies suggest that both DHPG and HPMPC may have inhibitory effects on the development of certain immune functions at high dosages, but at drug concentrations that were therapeutic in animal model studies, little inhibitory effects were observed.

Correlation of growth of tumours in NC-cell-depleted mice with NC- and NK-cell-mediated lysis in vitro.

The anti-tumour surveillance activity of natural cytotoxic (NC) cells was studied in vivo using the transplantable tumours WEHI-164 fibrosarcoma, MPC-II plasmacytoma, WEHI-7 T-lymphoma, B16 melanoma and EL-4 thymoma in syngeneic and semi-allogeneic mice. Experimentally, mice were treated with the anti-NC-I.I monoclonal antibody (MAb) IC4 to abrogate splenic NC activity. This was followed by s.c. inoculation of MTD100 doses of the tumours. Comparison of the diameters of the tumours in the anti-NC-I.I-treated mice with control mice using non-parametric statistics showed significantly faster growth of WEHI-164 (p less than 0.01), MPC-II (p less than 0.05) and WEHI-7 (p less than 0.05) when the mean tumour diameters were less than 15 mm in the anti-NC-I.I-treated mice. Significantly faster growth was also observed in anti-NC-I.I-treated mice with the B16 tumour (p less than 0.05), but at a later stage of growth, when the tumour diameter was greater than 15 mm. In vitro, WEHI-164, MPC-II and WEHI-7 were shown to be predominantly sensitive to lysis by mouse splenic NC cells, while B16 was predominantly lysed by splenic natural-killer (NK) cells. Anti-NC-I.I treatment of mice did not affect the growth of EL-4 in vivo and in vitro experiments with anti-NK-I.I and anti-NC-I.I MAb indicated that this tumour was lysed by sub-sets of NK and NC cells distinct from those which lysed the other tumours. We conclude that, in mice at least, NC cells have an in vivo role in controlling the growth of some transplantable tumours, and this correlates with the in vitro NC cell lysis of these same tumours.

Alteration in mouse splenic phospholipid fatty acid composition and lymphoid cell populations by dietary fat.

The fatty acid composition of diacyl- and alkylacylglycerophosphocholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), alkenylacyl-glycerophosphoethanolamine (aPE), and diacyl- and alkylacyl-glycerophosphoethanolamine (dPE) was assessed in isolated splenocytes from C3H/Hen mice fed one of four purified isocaloric diets for six weeks. Diets contained 20% by weight of either a high-linoleate sunflower oil (Hi 18:2), a high-oleate sunflower oil (Hi 18:1), a mixture of 17% menhaden fish oil and 3% high-linoleate sunflower oil (Hi n-3), or a mixture of 17% coconut oil and 3% high-linoleate sunflower oil (Hi SFA). Spleen weight and immune cell yield were significantly higher (P less than 0.05) in mice fed the Hi 18:1 or the Hi n-3 diets compared with those fed the Hi 18:2 and Hi SFA diets. Distinctive patterns of fatty acids were observed for each phospholipid in response to dietary fatty acids. Dietary fat significantly affected (P less than 0.05) total polyunsaturated fatty acids (PUFA) in PC and dPE, total saturated fatty acids (SFA) in PC, total monounsaturated fatty acids (MUFA), and n-3 PUFA in all phospholipid classes examined. In mice fed the Hi n-3 diet, n-3 PUFA were significantly elevated, whereas n-6 PUFA decreased in all of the phospholipids. In these mice, eicosapentaenoic acid (EPA) was the predominant n-3 PUFA in PC and PI, whereas docosahexaenoic acid (DHA) was the major n-3 PUFA in aPE and PS.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methylindole-induced splenotoxicity: Functional analysis of immune parameters and lymphocyte phenotyping by flow cytometry

In this laboratory, 3-methylindole (3-MI), a pneumotoxic metabolite of l-tryptophan that forms in the digestive tract of humans and ruminants, has been demonstrated to be toxic to rat and mouse splenic cells both in vitro and in vivo. The present studies examine whether the reduction in nucleated splenic cells is associated with alterations in: (1) immune functioning (e.g., B and T cell mitogenic responses to lectins), (2) natural resistance (e.g., natural killer (NK) activity and cytokine release from macrophages (MPs)), or (3) the relative percentages of B and T cells in the remaining cells as determined by flow cytometric phenotyping. A dose-dependent decrease in splenic weight (24–46%) and nucleated cell numbers (54–73%) was observed 24 hr after intraperitoneal (ip) administration of 100–300 mg/kg 3-MI to B6C3F1 mice. At a dose of 300 mg/kg, the blastogenic response of splenic lymphocytes to 1 μg/ml phytohemagglutinin, a T cell mitogen, was reduced 37 and 64%, and NK activity was reduced 20 and 60%, in rats and mice, respectively. Following exposure to 400 mg/kg 3-MI, interleukin-1 and tumor necrosis factor production by lipopolysaccharide-stimulated rat splenic MPs was decreased 58 and 38%, respectively. Despite the reduction in total nucleated cell number in 3-MI-treated mice, the percentages of splenic B and T cells remained the same. These findings indicate that, in addition to its toxicity to splenic cells, 3-MI can significantly impair the functioning of the remaining viable cells. The potential importance of these functional changes for alterations in host resistance in rodents exposed to 3-MI or other alkylindoles is unknown.

3-Methylindole-induced splenotoxicity: Biochemical mechanisms of cytotoxicity

3-Methylindole (3-MI) is a pneumotoxic metabolite of l-tryptophan that can form in the digestive tracts of humans and ruminants as a result of microbial protein metabolism. Alternatively, human lungs can be directly exposed to 3-MI formed during protein pyrolysis and inhalation of tobacco smoke. 3-MI has been shown to cause acute lung injury in both ruminants and rodents. The present studies demonstrate that the spleen is also a target for 3-MI-induced toxicity. A dose-dependent decrease in splenic weight (24–75%) and nucleated splenic cell number (22–68%) was observed 24 hr after intraperitoneal injection of 3-MI (50–300 mg/kg) to intact and adrenalectomized rats. These findings were associated with significant alterations in splenic histopathology. Mice appeared less affected by 3-MI than rats as no splenotoxicity was observed at doses less than 200 mg/kg. Other mono- and dimethyl-substituted indoles did not decrease mouse spleen cell numbers when administered in vivo. Phenobarbital pretreatment in vivo protected against 3-MI-induced splenotoxicity, suggesting a role for cytochrome P450-mediated metabolism of 3-MI in the splenotoxicity of this compound. Exposure of rat or mouse splenic cells to 3-MI (1 m m) in vitro resulted in toxic changes over 24 hr. However, equimolar concentrations of the structurally related mono- and dimethylindoles were also toxic in vitro, and preincubation with a variety of inhibitors of cytochrome P450 or prostaglandin synthase in vitro failed to protect against 3-MI-mediated toxicity to splenic cells in culture. These results suggest mechanisms of 3-MI splenotoxicity also exist that do not require bioactivation, and indicate a possible role for alkylindoles in suppression of immune function.

Suppression of lymphocyte proliferation through the nitric oxide synthesizing pathway

The amino acid l-arginine can be metabolized through a nitric oxide-synthesizing pathway (NOSP) to produce l-citrulline and reactive nitrogen intermediates. Among these nitrogen intermediates, NO has been implicated as the mediator of a variety of biological effects including vasodilatation, inhibition of platelet aggregation, tumor cytotoxicity and microbiostasis by activated macrophages and generalized suppression of macrophage functions. Work reported here demonstrated that the NOSP is expressed in Con A-stimulated rat splenic cell (SC) cultures and is associated with a profound suppression of lymphocyte proliferation. Inhibition of the NOSP by N G-monomethyl- l-arginine ( N-MMA) or binding of its products by hemoglobin, either free in solution or contained in RBC, markedly promotes rat SC mitogenic response to Con A. Mouse SC do not express the NOSP under the conditions used in these experiments. Consequently, their mitogenic response to Con A is not affected by N-MMA or hemoglobin. These data confirm and expand the apparent role of NO as a regulator of immune responses while indicating potentially important species differences.