Cell Blastogenesis Research Articles

Characterization of early IL-12, IFN-alphabeta, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection.

Murine cytomegalovirus (MCMV) infection of mice induces early cytokines. Although certain of these can directly inhibit viral replication, they also can promote defense by activating NK cells. MCMV induces IFN-alphabeta-dependent NK cell cytotoxicity and IL-12-dependent NK cell IFN-gamma production. Studies were initiated to define cytokine-mediated NK and T cell-independent antiviral defense and specific cytokine-elicited NK cell responses during MCMV infections. IFN-alphabeta, TNF, IL-12, and IFN-gamma were all shown to be induced 2 days after infection of immunocompetent mice. Infections of NK and T cell-deficient mice demonstrated that virus-induced IFN-alphabeta, TNF, and IL-12, but not IFN-gamma, were produced independently of these populations, and that IL-12 production occurred in the absence of detectable IFN-gamma. In vivo neutralization studies of IFN-alphabeta, TNF, and IL-12 showed that each of these factors had NK and T cell-independent antiviral functions, as well as specific effects on NK cell responses. Examination of NK cell cytotoxicity, blastogenesis, and IFN-gamma production demonstrated that: IL-12 was required for NK cell IFN-gamma production but not blastogenesis and cytotoxicity; IFN-alphabeta was necessary for NK cell blastogenesis and cytotoxicity but not IFN-gamma production; and TNF facilitated IFN-gamma production but inhibited NK cell cytotoxicity. This work defines the biologic consequences of early cytokine expression during viral infection.

CD40-mediated stimulation contributes to lymphocyte proliferation, antibody production, eosinophilia, and mastocytosis during an in vivo type 2 response, but is not required for T cell IL-4 production.

CD40/CD40 ligand interactions are required for the development of T cell-dependent Ab responses in vivo. The role of these cell surface molecules in contributing to T cell cytokine production and the development of effector populations other than B cells and T cells is, however, less well defined. We have examined the in vivo effects of blocking CD40/CD40 ligand interactions on the type 2 mucosal immune response that follows oral inoculation of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of anti-gp39 (CD40L) mAb (MR1) blocked H. polygyrus-induced elevations in serum IgG1 levels and inhibited elevations in blood eosinophils and mucosal mast cells at day 14 after inoculation. Anti-gp39 mAb markedly inhibited B cell blastogenesis 8 days after H. polygyrus inoculation but did not inhibit elevations in B cell class II MHC expression. Maximal elevations in B7-2 expression required signaling through both CD40 and the IL-4R. Elevations in T cell cytokine gene expression and elevations in the number of IL-4-secreting cells were unaffected by treatment with anti-gp39 mAb, although IL-4 production was inhibited by anti-IL-4R mAb. These results suggest that CD40/CD40L interactions are not required to activate T cells to produce cytokines but are required for the activation and proliferation of other effector cells associated with the type 2 response.

Function and Regulation of Natural Killer (NK) Cells during Viral Infections: Characterization of Responsesin Vivo

Althoughin vitrosystems have provided important information about the composition and nature of various immune responses, understanding physiologically relevant function and regulation requires evaluatingin vivoconditions. Two different models of acute viral infections have made possible the characterization of a variety of responses to these agents, including natural killer (NK) cell activation and regulation during infection; these are mouse infections with lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). The results of our characterization of the NK cell responses elicited in these models and the methods used to dissect the regulation of these responses are reviewed here. Cytotoxicity, proliferation, and cytokine expression assays as well as flow cytometric analyses are used to characterize thein vivoresponses. Both of the infections induce early NK cell cytotoxicity and blastogenesis. Infection with MCMV but not LCMV also induces NK cell production of the antiviral cytokine, interferon-γ (IFN-γ). Antibodies, to mediatein vivocell subset depletion or cytokine neutralization, and mice, genetically altered to have cell subset or cytokine deficiencies, are utilized to identify the regulatory pathways and mechanisms controlling endogenous NK cell responses to the infections. The major mediators of the regulation of NK cell function during viral infection of normal mice are IFN-α/β and interleukin-12 but not interleukin-2. Furthermore, the induction of later T-cell responses contributes to the negative regulation of NK cells by promoting the production of inhibitory factors including biologically active transforming growth factors-β. Thus, the study of immune responses to viral infections has provided and will continue to provide important insights into the characteristics of endogenous NK cell responses and the cells and factors that regulate these responsesin vivo.

Immunomodulatory activity of blood serum from chromium-supplemented periparturient dairy cows

Our previous research showed enhanced immune responses, including mitogen-induced blastogenesis of peripheral blood mononuclear cells from feedlot calves and periparturient dairy cows supplemented with dietary chromium (Cr). The objectives of the present study were to test whether blood sera from Cr-supplemented periparturient cows contained immunomodulatory activity for mitogen-stimulated peripheral blood mononuclear cells and, if so, to determine if this activity was explicable by differences in blood profiles of some glucose-regulating hormones (insulin, cortisol, growth hormone, insulin-like growth factor I, and tumor necrosis factor-α) between Cr-supplemented and unsupplemented (control) animals. Blood sera from ten unsupplemented cows and nine Cr-supplemented cows (0.5 ppm day −1) were collected weekly from 2 weeks before to 6 weeks after parturition, and were used to supplement (1, 10, and 20% vol/vol) culture medium supporting concanavalin A (Con A) stimulated mononuclear cells enriched from blood of four nulliparous donor cows. Hormone concentrations were determined using radioimmunoassays. Con A-induced blastogenesis was enhanced when 1, 10, and 20% sera from Cr-supplemented cows was added to the mononuclear cell cultures, and this was particularly evident around parturition. Conversely, peripartum sera from unsupplemented cows depressed Con A-induced blastogenesis. Except for a marginal rise in blood cortisol 2–4 weeks after parturition, no significant effects of Cr supplementation on other hormones (insulin, growth hormone, insulin-like growth factor I, tumor necrosis factor-α) were observed. These observations suggest that factors in peripheral blood serum from Cr-supplemented cows, other than absolute concentrations of the glucose-regulating hormones studied, modulate Con A-induced blastogenesis of mononuclear leukocytes.

Effects of enalapril on T and B cell function in rats after myocardial infarction

The cellular mechanisms following myocardial infarction remain poorly characterized. It is believed that an inflammatory and immunologic process may be involved and that the beneficial effects of enalapril on remodeling may, in part, work through an immune mechanism. To characterize the effect of enalapril on immune alterations in the late phase of ventricular remodeling after myocardial infarction, rats underwent left coronary artery ligation followed by 6 weeks of either enalapril or placebo treatment. Infarct sizes, heart weights, and volumes were compared. Peripheral and splenic leukocyte and lymphocyte subsets, along with T cell blastogenesis, were quantified in enalapril treated rats 6 weeks after coronary ligation and compared to untreated control rats. Additionally, antibody production to a de novo antigen, keyhole limpet hemocyanin, was assessed with and without treatment. Average infarct size was equivalent among enalapril-treated myocardial infarction rats and untreated infarct rats. There was, however, less left and right ventricular hypertrophy in the enalapril treated group. Enalapril completely prevented the 42% increase in white blood count, the 88% increase in neutrophils, and the 28% increase in lymphocyte count seen in untreated infarct rats. Both untreated and enalapril treated rats tended toward a decrease in T helper:suppressor ratio. All rats treated with enalapril, however, had a significant increase in the T helper:suppressor ratio versus untreated control rats (F = 3.6, P = .018). Blastogenesis was markedly increased in T cells from infarcted animals. This was mitigated by treatment with enalapril. Additionally, immunoglobulin G antibody production was significantly lessened in rats treated with enalapril. The results of this study suggest that alterations in immunoregulatory cell type and function occurs following myocardial infarction. The beneficial effects of the angiotensin-converting enzyme inhibitor enalapril may be, in part, due to its mitigating effects on immune cell release and activation following myocardial infarction.

Studies on the phenotypic and functional characterization of peripheral blood lymphocytes from patients with early-onset periodontitis.

Juvenile and rapidly progressive periodontitis are grouped under the heading of patients with early-onset periodontitis (EOP). Many studies have investigated host risk factors in the etiology of EOP patients but these remain inconclusive. This study was undertaken to assess the possibility that an abnormality in the systemic lymphocyte subpopulation or function is involved in the etiology of EOP patients. Fourteen (14) patients with juvenile periodontitis (JP), 18 with rapidly progressive periodontitis (RPP), 22 with adult periodontitis (AP), and 33 with a healthy periodontium (HP) participated in this study. Lymphocyte subsets were determined by using panels of monoclonal antibodies (mAbs) and fluorescent flow cytometry. T cell blastogenesis was evaluated by [3H]-thymidine uptake. Pokeweed mitogen induced immunoglobulin G (IgG) and IgM synthesis were detected by sandwich enzyme-linked immunosorbent assay. There were wide distributions of values in all examinations among subjects. No significant difference could be found between the periodontitis patients and HP groups with the exception of a high CD4/CD8 ratio in all patient groups (P < 0.0001) and the depressed percentages of CD3 positive cells noted in the AP patient group (P < 0.0001). These results suggest that the majority of EOP patients do not show significantly different lymphocyte profiles from AP patients and HP subjects, and that lymphocyte cell dysfunctions are not always seen, even in EOP patients.

Influence of aging and chemoradiotherapy on leucocyte function in oral cancer patients.

Leucocyte functions and the influence of chemoradiotherapy were examined in three age groups of patients with oral cancer. The groups consisted of 66 patients below 65 years old (group A): 40 patients between 65 and 80 years old (group B); 20 patients over 80 years old (group C). 20 healthy individuals (45.8 +/- 9.6 years old) were chosen as controls. Originally, no significant differences in leucocyte count, CD3 population, CD4/CD8 ratio, natural killer activity or phagocytosis of polymorphonuclear leucocytes (PMNL) were found in the patients. However, T cell blastogenesis, lymphokine-activated killer cell activity and superoxide production of PMNL were all suppressed. These functions were further suppressed by cancer therapy, the greatest suppression being seen in group C. Compared to controls and group A, the generation of interleukin-1, interleukin-6, tumour necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor were markedly suppressed in group C. These results show that very old cancer patients are already in an immunologically suppressed condition and that the leucocyte functions of these patients are further impaired by cancer therapy.

2-Acetylaminofluorene inhibits the activation of immune responses by blocking cell cycle progression at G1 phase.

2-Acetylaminofluorene (AAF) inhibited in a dose dependent manner mouse spleen cell blastogenesis in response to phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Io) activation, the T-cell lectin, concanavalin A (Con A), and following stimulation by alloantigens as measured by the mixed lymphocyte response (MLR). AAF also markedly suppressed the T-cell dependent antibody forming cell (AFC) response to sRBC. AAF was most inhibitory on both the sRBC IgM AFC response and Con A stimulated proliferation when added during the first 24 h following initiation of culture. Direct addition of high concentrations of AAF (100 microM) to spleen cell cultures at 48 h following Con A stimulation produced a very modest inhibition (< 20%) of T-cell proliferation as compared to 90% when added at the time cultures were initiated. Similarly, AAF (75 and 100 microM) produced a greater than 80% inhibition of the in vitro AFC response when spleen cells were sensitized with antigen in presence of AAF. In contrast, no inhibition of the IgM AFC response was produced when AAF (75 microM) was added to spleen cell cultures 48 or 72 h after antigen sensitization. Con A-triggered cell-cycle progression was attenuated at the G1 stage by the addition of AAF (50 and 100 microM) with no inhibition of S to G2/M phase transition. These results suggest that the mechanism of AAF-mediated immune suppression is through a blockade of cell cycle progression from G1 to S phase.

Influence of organophosphorus compounds on different cellular immune functions in vitro

The effects of two organophosphorus compounds (OP) triphenyl phosphate (TPP) and triphenylphosphine oxide (TPPO) on different immune functions in vitro were investigated using an economical multiple endpoint approach. The test battery was designed as follows: immunocompetent cells (peritoneal cells and splenocytes) were isolated from female C57B1 mice and treated in vitro for 1 hr with the test article. Then the cells were washed and assessed for the following immune functions: Fc-receptor-dependent phagocytosis and lipopolysaccharide-induced release of a cytolytic protein (tumour necrosis factor, TNF) of thioglycollate-elicited peritoneal macrophages; natural killer (NK) cell activity, blastogenesis (T and B lymphocytes), and antibody synthesis (B lymphocytes) of spleen-cell suspensions. As an additional parameter the survival of the cells was determined routinely after treatment during all assays. No substantial effect on macrophage phagocytotic activity was observed after TPP or TPPO treatment. TPP led to a concentration-dependent suppression of macrophage TNF activity as well as NK activity of spleen cells. In addition, a slight reduction of B-lymphocyte antibody synthesis was obtained. TPPO treatment revealed a modulation of TNF activity of macrophages in a complex, non-concentration-related manner. A concentration-related suppression of spleen cell NK activity was observed after TPPO treatment. In summary, TPP and TPPO were found to be immunomodulating agents eliciting adverse effects predominantly towards cells of the innate immunity. The functions of T and B lymphocytes, referred to as adaptive immunity, were not substantially impaired. Our results indicate that the in vitro test battery described may be a suitable tool for the screening of OP-mediating immunotoxicity.

Murine B cell proliferation and protection from apoptosis with an antibody against a 105-kD molecule: unresponsiveness of X-linked immunodeficient B cells.

We established a novel monoclonal antibody, RP/14, that can protect B cells from apoptosis induced by irradiation or dexamethasone. A molecule recognized by RP/14 (the RP antigen) was expressed on B cells with B220bright, IgMdull, and IgDbright. Immunoprecipitation experiments revealed that RP/14 recognized a monomeric protein with an approximate molecular mass of 105 kD. Stimulation of B cells with RP/14 for 48 h induced B cell proliferation and blastogenesis. In contrast to B cells of wild-type mice, X-linked immunodeficient (XID) B cells did not proliferate upon stimulation with RP/14, although the RP antigen was expressed to the same extent as that of wild-type B cells. These results suggest that the RP antigen-mediated signaling pathway is important for rescuing B cells from apoptosis and is deficient in XID B cells.

Role of eicosanoids and vitamin E in fish oil-induced changes of splenocyte proliferation to T cell mitogens in mice

The effect of (n-3) polyunsaturated fatty acids (PUFA)-enriched diets on T cell blastogenesis and the contributions of (n-3) PUFA-derived eicosanoids and tocopherol to this effect were investigated in C57BL/6NIA mice. Splenocytes from male, 2 month old mice (n=24/group) fed diets enriched in either (n-3) or (n-6) PUFA (10 g/100g diet) for 8 weeks were activated with T-cell mitogens and cultured with and without varying concentrations of PGE 2, PGE 3, LTB 4, LTB 5, vitamin E or indomethacin. There was a significant decrease in the percentage of T cells, CD4+, CD8+ and macrophages in spleens from the (n-3) PUFA-fed group. Phytohemagglutinin (PHA) response was significantly suppressed (−15%) in splenocytes from the (n-3) PUFA-fed group. In vitro addition of PGE 2 or PGE 3 at both 10 −7 M and 10 −8 M significantly suppressed PHA-activated proliferation in the (n-6) PUFA-fed group (p<0.01). In the (n-3) PUFA-fed group, however, PHA response was suppressed with the addition of 10 −7 M PGE 2. or PGE 3 only (p<0.01). Although PGE 2 had no significant effect on concanavalin A (Con A) activation of splenocytes from either dietary group, PGE 3 (10 −7 M) significantly suppressed Con A activation of splenocytes from both dietary groups (−21±8% in the (n-6) PUFA group vs −24±7% in the (n-3) PUFA group; p<0.05). The addition of LTB 5 at 10 −10 to 10 −12 M to cultures from the (n-3) PUFA group resulted in a weak suppression of Con A-induced response while no effect in the (n-6) PUFA diet group was observed. Vitamin E significantly enhanced proliferation by 281% in the (n-3) PUFA-fed group (p<0.01) and 109% in the (n-6) PUFA-fed group (p<0.02). Cyclooxygenase inhibition caused a significant increase in mitogenic response of splenocytes from the (n-3) PUFA group while it had no effect on that of the (n-6) PUFA group. It is concluded that EPA-derived eicosanoids as well as changes in vitamin E requirement contribute to the (n-3) PUFA-induced changes in lymphocyte proliferation.

Differential modulation of chicken lymphocyte blastogenesis and cytotoxic activity of natural killer cells in vitro by retinol, retinoic acid and beta-carotene

Summary The effects of retinol (ROL), retinoic acid (RA) and B-carotene (CAR) on the response of chicken lymphocyte blastogenesis and on the cytotoxic activity of natural killer (NK) cells were evaluated in vitro. Speenocytes were isolated from healthy growing chicks and cultured in presence of different concentrations of ROL, RA and CAR under similar experimental conditions. Lymphocyte response to concanavalin A (Con A) was affected by adding ROL, RA and CAR to the cultures, by the time of exposure and by methods used for diluting ROL, RA and CAR stock solutions. Addition of ROL stock solutions diluted in cell culture medium suppressed lymphocyte response in a linear dose-dependent manner whereas pre-dilution of ROL stock solutions in fetal bovine serum (ROL-FBS) increased blastogenesis in a linear manner. Addition of 10 μM of ROL-FBS at the beginning of the incubation period or 16 h after enhanced proliferative responses by 5 and 9 times, respectively, as compared to Con A-induced cells without ROL. Suppressed proliferative response of Con A-induced cells was seen when both RA and CAR working solutions were added at a concentration of 10 μM and 1 μM, respectively. However, RA and CAR pre-diluted in FBS (RA-FBS, CAR-FBS) increased proliferative response of Con A-induced cells in a quadratic dose-dependent manner. At peak levels of RA-FBS and CAR-FBS, blastogenesis was respectively 2.1 and 1.3 times higher than activated cells without RA-FBS or CAR-FBS. No significant increase was detected with RA or CAR directly diluted in RPMi medium. Cytotoxic activity of chicken NK cells was reduced when ROL was added to the cultures, whereas RA and CAR enhanced NK activity. Both preparations of tested compounds had similar effects on NK activity. Our results showed that immunocompetent cell functions were modulated by retinol, retinoic acid and B-carotene in a dose-dependent manner and by the method used to prepare the working solutions.

Resting and anergic B cells are defective in CD28-dependent costimulation of naive CD4+ T cells.

Successful antibody production in vivo depends on a number of cellular events, one of the most important of these being cognate B cell-T cell interaction. To examine this phenomenon in vitro, homogeneous populations of hen egg lysozyme (HEL)-specific small resting B cells and naive CD4+ HEL-specific T cells (derived from immunoglobulin [Ig] and T cell receptor transgenic mice, respectively) were cultured together. On addition of intact HEL protein. HEL-specific B cells increase their expression of activation molecules, including a B7-related protein and CD44, and enlarge into blast cells. Within the same cultures, HEL-specific CD4+ T cells also increase expression of the activation markers CD69 and CD44, enlarge, secrete lymphokines, and proliferate. This response is radiation sensitive, supporting the conclusion that HEL-specific B cells present antigen to and activate the naive T cells. By contrast, when a synthetic peptide fragment of HEL is used to bypass B cell antigen-receptor engagement, the naive T cells enlarge and display activation antigens, but fail to produce lymphokines, proliferate, or promote B cell blastogenesis. Presentation of HEL by tolerant B cells, which are no longer able to signal effectively through their antigen receptors, results in an identical pattern of incomplete T cell activation. Addition of a stimulating anti-CD28 antibody and blocking of CD28 signals with CTLA4/Ig fusion protein both show that complete activation of naive CD4+ T cells depends on the initial induction of B7 and related costimulatory molecules after HEL binding to nontolerant HEL-specific B cells. Thus, in the absence of adequate constimulation from the B cell, naive CD4+ T cells undergo a form of "partial activation" in which they upregulate surface expression of certain T cell activation antigens, but fail to efficiently produce lymphokine and proliferate. This may explain the different conclusions that have been reached regarding the consequences of B cell antigen presentation to T cells, in that the ability of B cells to activate naive CD4+ T cells depends both on their specificity and their activation state.

Cellular Interactions of Murine Immune Cells Exposed to Live Mycobacterium intracellulare, Its Whole Lipid Extract, and Its Serovar-Specific Glycopeptidolipid

In this study we examined some of the immunological responses to Mycobacterium intracellulare and its lipid components. Our results indicate that infection with M. intracellulare can increase the expression of adhesion molecules, ICAM-1 and LFA-1, only at the site of injection (peritoneum). There was no change in the expression of these adhesion molecules in the lymphoid organs (thymus and spleen). Significant increases in the adhesion molecules were observed in the spleen cells incubated with the lipid derived from mycobacteria in the presence of concanavalin (Con A) compared to the Con A alone. The expression of the Thy 1.2 and Lyt-2 markers was not affected by the bacteria or their lipids. The results indicate a marked increase in the mitogenic response by the infected spleen cells removed at an early day. The blastogenic study also indicated that the lipids can reduce the mitogen-induced blastogenesis of spleen cells removed from M. intracellular- and saline-injected mice; moreover, they suggested that the spleen cells removed from Listeria monocytogens-infected mice can also be affected by mycobacterial lipids. This indicates a nonspecific effect by these lipids. The results that the immunological response was contingent upon prior exposure of the mice to M. intracellulare, and also was dependent on whether the cells came from the peritoneal cavity or lymphoid organs.

Integrin alpha 4 beta 7 co-stimulation of human peripheral blood T cell proliferation.

The integrin alpha 4 beta 7 mediates lymphocytes adhesion to VCAM-1 on activated endothelium, fibronectin in the extracellular matrix, and the mucosal vascular addressin MAdCAM-1. It is unclear whether alpha 4 beta 7 performs any function beyond directing specific adhesion reactions. We addressed the possibility that triggering of alpha 4 beta 7 with a specific monoclonal antibody was capable of delivering an accessory stimulus that would coactivate T cells and lead to proliferation. At submitogenic levels of anti-CD3 stimulation, triggering of alpha 4 beta 7 by immobilized mAb ACT-1 resulted in T cell blastogenesis, IL-2 production, expression of the IL-2 receptor alpha chain CD25, and ultimately DNA synthesis. These results indicate that the integrin alpha 4 beta 7 is involved in more than lymphocyte adhesion and homing but also plays a role in cell signaling.

Defective leukocyte function-related cryoprecipitable protein in cryoglobulinemia.

Cryoprecipitable proteins (CPP), purified from plasmas removed during plasmapheresis treatment of patients with type II and type III cryoglobulinemia (CG), were evaluated in vitro for their effect on normal leukocyte function. CPP reduced normal mononuclear cell blastogenesis and polymorphonuclear cell phagocytosis responses in a dose-dependent fashion. At the maximum concentration studied, 5,000 micrograms/ml, CPP were shown to suppress normal blastogenesis and phagocytosis 30-60% and 50-80% below the albumin control, respectively. A 50% suppression of normal blastogenesis and phagocytosis was observed at 500 micrograms/ml for type III CPP. Leukocyte functions in CG patients were evaluated and compared to those of healthy controls. Blastogenesis and phagocytosis were significantly inhibited in CG patients. These studies suggest that CG patients have defective leukocyte function and that CPP have suppressive effects on normal leukocyte function. Our studies suggest that removal of CPP in CG patients may prove beneficial not only in reducing typical symptoms of CG but also in reducing the potential inhibitory effects of CPP on leukocyte function.

Effects of supplemental chromium on immune responses of periparturient and early lactation dairy cows.

The objective of the present study was to determine the effects of supplemental dietary chromium on immune responses of dairy cows subjected to physical and metabolic stresses associated with late pregnancy, calving, early lactation, and peak milk yield. Nine periparturient dairy cows were supplemented with chelated Cr (.5 ppm/d) from 6 wk prepartum (wk -6) through 16 wk postpartum (wk 16), and 10 cows were unsupplemented controls. To assess humoral immune responses, all cows were immunized with ovalbumin (OVA; s.c.) and human erythrocytes (HRBC; i.v.) on wk -2 and 2, and sera from weekly blood samples were assayed for content of antigen-specific antibody. Cell-mediated immunity was assessed in vitro using antigen (OVA)- and mitogen-stimulated peripheral blood mononuclear cell (PBMC) blastogenesis of cells collected biweekly from wk -2 and 6. Supplemental Cr caused anti-OVA antibody responses (P < .01) and mitogen-stimulated blastogenic responses of PBMC (P = .05) to be elevated, was associated with lowered OVA-stimulated blastogenic responses of PBMC (P < .01), and had no overall effect on antibody responses to HRBC (P > .10) relative to responses of control cows. These results confirmed and extended our previous observations that supplemental Cr can alter specific immune responses of stressed cattle.

Suppression by HIV of IL-1 and IL-6 Secretion in Accessory Cells: AC Function Defect Partially Corrected with Exogenous IL-1 and IL-6

To determine the effect of HIV infection on the accessory cell function of monocytes we measured the ability of HIV-infected monocytes to restore PHA-induced and soluble anti-CD3-induced T cell blastogenesis. These T cells were highly purified and depleted of monocytes (<0.5%) and activated T cells. Monocytes were isolated using gelatin-fibronectin-coated flasks (<1% T cells) and after 4 days in culture with granulocyte/macrophage-colony stimulating factor, they were infected with HIV. Accessory cell (AC) function was tested 2 and 7 days later, employing autologous cryopreserved T lymphocytes. Monocytes infected with HIV for 2 days lacked the ability to permit phytohemagglutinin (PHA) and anti-CD3-induced T cell blastogenesis. Noninfected monocytes restored the proliferative response of purified T cells. Interleukin 1 (IL-1) and interleukin 6 (IL-6) levels in culture supernatants were low when compared to cultures with noninfected AC. Preincubation of monocytes with human anti-HIV neutralizing antibodies did not restore either of the responses. AC treated with heat-inactivated HIV had normal accessory cell function. The addition of IL-1 and/or IL-6 partially restored the AC function for PHA stimulation, but not for anti-CD3 stimulation. We conclude that HIV infection of monocytes suppressed their accessory cell function in the T cell blastogenesis assay. The response was partially restored with IL-1 and/or IL-6, suggesting that HIV infection down-regulated the monocyte production of both cytokines.

Differential induction of prostaglandin E2-dependent and -independent immune suppressor cells by tumor-derived GM-CSF and M-CSF.

Both prostaglandin E2 (PGE2)-dependent (indomethacin-sensitive) and PGE2-independent (indomethacin-insensitive) suppressor cell activities that inhibited mitogenic T cell blastogenesis appeared in the bone marrow and spleen of mice on days 20 to 30 following transplantation of NFSA fibrosarcoma molecularly expressing mRNA for both macrophage (M) and granulocyte-macrophage (GM) colony-stimulating factors (CSFs). The present study was done to characterize the two different suppressor cells isolated from NFSA tumor-bearing mice and to verify a role of CSFs in the induction of suppressor cells in vitro. Whereas PGE2-releasing suppressor cells were found in bone marrow and spleen cells isolated from tumor-bearing mice, indomethacin-insensitive suppressor cells in both tissues were localized predominantly in adherent cell fractions. An increase in Mac-1+ and Mac-2+ spleen cell populations with two to three times larger cell volumes was observed, and both showed strong PGE2-releasing capacity and indomethacin-sensitive suppressor cell activity. However, after elimination of Mac-1+ or Mac-2+ cells, bone marrow cells still showed higher PGE2-releasing capacity and indomethacin-sensitive suppressor activity. The in vitro cultures of normal bone marrow and spleen cells with NFSA cell conditioned medium (NFSA-CM) induced heterogeneous mixtures of indomethacin-sensitive and - insensitive suppressor cells like those observed in cultures with the combination of M-CSF and GM-CSF. However, cultures with either GM-CSF or M-CSF resulted in the induction of indomethacin-sensitive suppressor cells by GM-CSF and of indomethacin-insensitive suppressor cells by M-CSF. In addition, NFSA-CM pretreated with anti-GM-CSF antibody induced indomethacin-insensitive suppressor cells in in vitro cultures of bone marrow and spleen cells. These results suggest that two distinctly different suppressor cells developed under hemopoiesis of myelomonocytic lineage cells are regulated differentially by the two macrophage growth factors, M-CSF and GM-CSF.

A modified method of mixed lymphocyte reaction: establishment of the assay system and its application to extracts of fungal cultures.

A modified mixed lymphocyte reaction (MLR) assay was developed to screen immunosuppressive agents. In the MLR, irradiation of splenocytes with UV light was employed for the preparation of stimulator cells, and the highest response was observed with the combination of splenocytes of C3H/He mice as a stimulator and those of C57BL/6 as a responder. The blastogenesis of the responder cells was quantified by a colorimetric method using MTT in 96-well microculture plates. For screening immunosuppressive substances, the MLR in combination with a cytotoxicity test can distinguish immunosuppressors from cytotoxic agents. The applications of this assay system to extracts of fungal culture was also described.