Antibody-forming Cell Response Research Articles

Modulation of antibody-forming cell and mitogen-driven lymphoproliferative responses by dietary nucleotides in mice

Several studies have demonstrated that dietary nucleotides play a role in maintaining T-cell dependent immunity. In this work, we investigated the effects of nucleotide supplementation of a nucleotide-free diet (NFD) on some immunity parameters in BALB c mice. Twenty day old mice were maintained on diets for 30 days prior to use in experiments. The addition of nucleotide mixtures to NFD resulted in an increase in the response of hemolytic IgG-forming cells induced by previous immunization with sheep erythrocytes. When NFD was supplemented with single nucleotides, AMP, GMP, or UMP increased the IgG response, whereas CMP and IMP were without effect. GMP was the only nucleotide that increased the hemolytic IgM-forming cell response. Neither the contact hypersensitivity response to dinitrofluorobenzene nor the time of death after transplantation of a syngenic lymphoma was modified by nucleotide addition to NFD. The in vitro proliferative response of splenocytes to LPS was not affected by nucleotide supplementation of NFD, but the ConA-driven proliferative response was increased in mice fed NFD supplemented with nucleotide mixtures or with UMP. These data show that dietary mononucleotides stimulated at least some T-cell dependent immunity mechanisms. Moreover, these stimulatory effects may be obtained by supplementing a nucleotide-free diet with a mixture in which mononucleotides are at the same levels as in murine breast milk.

IFN-alpha-mediated suppression of low-affinity FC(epsilon) receptors on Peyer's patch lymphocytes and augmentation of soluble CD23: implications for IgE responses.

The ability of interleukin (IL)-6 or interferon-alpha (IFN-alpha) to regulate expression of low-affinity Fc(epsilon) receptor (CD23) and serum levels of CD23 was studied in benzylpenicilloyl-keyhole limpet hemocyanin-sensitized BALB/c mice at the peak of a hapten-specific immunoglobulin E (IgE) antibody-forming cell (AFC) response. These responses are analogous to those observed in human atopic disease. To induce peak IgE responses, mice were injected intraperitoneally with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on days 0, 21, and 42. On day 44, mice were injected subcutaneously with IL-6 (100-1000 U) or IFN-alpha (1000-10,000 U). On day 46, numbers of CD23+ lymphocytes in Peyer's patches (PP), mesenteric lymph nodes (MLN), and spleen and levels of soluble CD23 in serum were determined (flow microfluorimetry and enzyme-linked immunosorbent assay, confirmed by competition assay). Data are expressed as percent total cells or as optical density at 490 nm. IFN-alpha treatment strongly suppressed (up to 100%) numbers of CD23+ cells exclusively in PP (i.e., numbers of CD23+ cells in MLN and spleen were unchanged) whereas serum levels of soluble CD23 were dramatically increased (60%). IL-6 treatment had no effect on either numbers of CD23+ lymphocytes or on serum levels of soluble CD23. The data suggest that the mechanism(s) by which IFN-alpha, but not IL-6, regulates IgE responses involves suppression of CD23 expression on lymphocytes in PPs and supports a central role for these organs in regulation of IgE responses in vivo.

Immune Function and Host Defense in Rodents Exposed to 60-Hz Magnetic Fields

This study was conducted to evaluate the influence of subchronic exposure to pure, linearly polarized 60-Hz magnetic fields (MF) on the host immune response in mice. The experimental design was as follows: three groups were exposed continuously (18.5 hr/day) to MF at field strengths of 0.02, 2, or 10 gauss (G), one group was exposed intermittently (1 hr on/1 hr off) to MF at a field strength of 10 G, and one group served as a sham control. Experimental endpoints included spleen and thymus weights and cellularity, antibody-forming cell (AFC) response, delayed-type hypersensitivity (DTH) response, splenic lymphocyte subset analysis, susceptibility to infection with Listeria monocytogenes, and natural killer (NK) cell activity. No differences in body weight, lymphoid organ weight, or lymphoid organ cellularity were observed in any MF-exposed group in comparison to sham controls. Likewise, no statistically significant differences were found in comparisons of AFC responses. Isolated statistically significant differences from control were observed in MF-exposed mice in the DTH assay, although no clear dose-related pattern of altered activity was seen. Splenic lymphocyte subset parameters examined were within normal limits in all groups, and no differences between control and MF-exposed mice were found. Host resistance to bacterial infection was not altered at any MF exposure examined in this study. Finally, although apparently dose-related, statistically significant alterations were observed in an initial study of NK cell function, repeat studies failed to demonstrate a consistent pattern of alteration.

Expression of functional glucagon receptors on lymphoid cells

The objective of the present studies was to determine whether the existence of functional glucagon receptors could be established on lymphoid cells. The glucagon receptor, which positively regulates adenylate cyclase, is a member of the superfamily of seven transmembrane domain G-protein coupled receptors. Previously reported specific binding with [ 125I]-glucagon to a variety of lymphoid and myeloid cell preparations suggests that glucagon receptors are expressed within the immune system. In the present study, Northern analysis of polyA RNA isolated from primary mouse and rat derived lymphoid tissues and lymphoid cell lines EL-4.IL-2, Jurkat E6-1, CH12LX, and BCL1-3B3 cells were probed with a 32P-labeled human hepatic glucagon receptor. Mouse spleen and thymus, rat spleen, and the B cell line, CH12LX, all possessed a single 1.5 kb fragment (BCL1-3B3, 1.4 kb) which hybridized to the glucagon receptor cDNA probe, as compared to mouse liver which exhibited a 2.8 kb fragment. EL-4.IL-2 and Jurkat E6-1 cells possessed a 3.7 kb fragment with an additional 2.75 kb band present in Jurkat E6-1 cells. Treatment of mouse splenocytes and T- and B-lymphoma cells with glucagon (0–100 nM) produced a dose-dependent enhancement in intracellular cAMP which was maximal at 5 min post treatment followed by a gradual decline. Direct addition of glucagon to spleen cell cultures over a broad concentration range produced no effect on either lymphoproliferation following stimulation with anti-CD3 mAb, or LPS nor on the antibody forming cell (AFC) response to sRBC. Conversely, glucagon effectively reversed the suppression of the sRBC AFC response produced by Δ 9-tetrahydocannabinol (Δ 9-THC), and partially reversed the suppression produced by 2′,3′-dideoxyadenosine, both of which are potent inhibitors of adenylate cyclase. These studies confirm the expression of functional glucagon receptors on lymphoid cells.

Development and Validation of the Antibody-Forming Cell Response as an Immunotoxicological Endpoint in the Guinea Pig

The mouse and rat are often the species of choice in standard toxicologj-cal testing. However, due to potential differences in chemical metabolism between these species and humans, they may in some instances be inappropriate models for chemical safety assessment. Because the guinea pig is a well-known model for chemical sensitization and hypersensitivity testing, the objective of the present study was to develop the hemolytic plaque assay in the guinea pig for use as an immunotoxicological endpoint in toxicology studies where metabolic differences between this and other rodent species may affect risk characterization. The present study encompassed six experiments designed to address route of administration, appropriate number of sheep red blood cells (sRBC) for immunization, time course after immunization, and a validation experiment to test the ability to detect suppression produced by a known immunosuppressant. Animals were observed daily for morbidity, mortality, and any signs of distress. Animals were weighed and euthanized and spleens were collected and weighed. The plaque assay was performed and the number of antibody-forming cells (AFQ/spleen, AFC/106 splenocytes, and total spleen cellularity was determined. The data indicate that retroorbital injection of 2 109 sRBC in a volume of 0.5 mL 5 days prior to euthanasia produces the peak AFC response in the guinea pig. In addition, IP injection of cyclophosphamide (30 mg/kg day−9) for 6 days prior to euthanasia produces profound immunosuppression in the absence of any observed overt toxicity. The peak serum anti-sRBC enzyme-linked immunosorbant assay (ELISA) response is delayed in comparison to the AFC response, and occurred on day 8 after retroorbital immunization in the guinea pig.

Effects of nicotine on the immune response. I. Chronic exposure to nicotine impairs antigen receptor-mediated signal transduction in lymphocytes.

Previous work has demonstrated that chronic exposure of rats to cigarette smoke causes inhibition of the antibody-forming cell (AFC) response and that the particulate phase of cigarette smoke, containing most of the nicotine in cigarette smoke, is essential for immunosuppression. Using intradermally implanted miniosmotic pumps, LEW rats were exposed to nicotine or its principal metabolite, cotinine, at the rate of about 14 micrograms/hr for 3-4 weeks. Serum cotinine levels in nicotine-treated (NT) animals of 219 +/- 40 ng/ml (on Day 10) were comparable to average human smokers. No significant differences between control (CON) and NT animals were observed in the distribution of lymphocyte subsets. However, nicotine, but not cotinine, treatment for 3 to 4 weeks inhibited both the T-dependent and T-independent AFC responses and proliferation to anti-CD3. Con A response was observed in 4-week but not in 3-week NT animals. Cell cycle analysis revealed that upon stimulation with Con A or anti-CD3, in spite of comparable surface expression of IL-2 receptors and class II MHC molecules, significantly fewer NT T cells entered the S and G2/M phases than CON T cells, indicating an arrest in the G0/G1 phase. Furthermore, B and T cells from NT animals were unable to elevate the intracellular calcium levels normally in response to ligation of antigen receptors, although Ca2+ responses of salivary gland cells to acetylcholine were normal. Thus, nicotine may significantly contribute to the immunosuppressive effects of chronic smoking by inducing a state of anergy in lymphocytes and may be related to their impaired response to antigen-induced signaling.

Use of Mishell-Dutton culture for the detection of the immunosuppressive effect of iron-containing compounds

Mishell-Dutton culture, known as an in vitro model for the evaluation of the humoral immune response of mice spleen cells to sheep red blood cells (SRBC), was used to study the immunosuppressive effect of iron-containing compounds. This response was indicated by the number of antibody forming cell (AFC) per million nucleated cells. Ferrous sulfate and ferric citrate (0.1 mM), when continuously present in Mishell-Dutton culture, significantly decreased the SRBC AFC response by approximately 63% and 86% of the control values, respectively. Ferric citrate, preincubated (24 h) with spleen cells and followed by lavage, significantly decreased the SRBC AFC response by approximately 54% of the control values. Primary and iron-treated coal, in concentrations ranging from 40 μg · ml −1 to 120 μg · ml −1, significantly decreased the SRBC AFC response when continuously present in Mishell-Dutton culture. Iron-treated coal, suppressed this response, in dose-dependent amounts, to a greater extent than did the primary coal: 73% versus 54% at 120 μg · ml −1. We concluded that Mishell-Dutton culture is suitable for studying the immunotoxicity of iron and these results may contribute to explain a decrease of host resistance against parasitic and bacterial infection in workers exposed to iron.

Local versus Systemic Immunotoxicity of Isobutyl Nitrite Following Subchronic Inhalation Exposure of Female B6C3F1 Mice

Local versus Systemic Immunotoxicity of Isobutyl Nitrite Following Subchronic Inhalation Exposure of Female B6C3F1 Mice. Ratajczak, H. V., Thomas, P. T., House, R. V., Gaworski, C. L., Sherwood, R. L., Luster, M. I., Hagen, K. L., Abdo, K., Jackson, C. D., Roycroft, J., and Aranyi, C. (1995). Fundam. Appl. Toxicol. 27, 177-184. Female B6C3F1 mice were exposed to isobutyl nitrite (IBN) by inhalation at 0, 37.5, 75, or 150 ppm for 6 hr per day, 5 days per week for 15 weeks. The potential of this compound to induce immunotoxicity was assessed during the 3rd, 13th, 14th, and 15th week of exposure and after 2 weeks of recovery following the 15 weeks of exposure. Both systemic and lung immune functions were examined, including body and lymphaid organ weights, pulmonary macrophage function and host defense, expression of splenic lymphocyte cell-surface markers, natural killer cell function, mixed lymphocyte reaction, and induction of specific antibody to a T-cell-dependent antigen. There was a dose-related suppression of T-cell-dependent antibody-forming cell responses in the spleen following IBN exposure; however, other measures of T-cell and nonspecific immunity were not significantly affected. A dose-related increase of H 2O 2 production by alveolar macrophages was present after 12 but not after 68 exposures to IBN. In contrast, pulmonary host defense mechanisms against Klebsiella pneumoniae were unaffected. These results suggest that in the absence of changes in host resistance, IBN may have selective and partially reversible effects on the immune system.

The B-cell response in lymphoid tissue of mice immunized with various antigenic forms of the influenza virus hemagglutinin

Protection of BALB/c (H-2d) mice against secondary challenge with influenza A viruses is primarily dependent on appropriate recognition of the hemagglutinin (HA) molecule by effectors of humoral immunity, the B lymphocytes and their product the immunoglobulin molecules. The influence of the antigenic form of the HA in eliciting protective antibodies is not clearly defined. We directly monitored the kinetics, character, localization, and helper T-cell dependence of the primary antibody-forming cell (AFC) response and the development of B-cell memory in lymphoid tissues associated with the upper and lower respiratory tracts, and in the spleen and bone marrow, to three forms of HA with various degrees of antigenic organization. Our results show that the antigenic organization of HA substantially influences B-cell immunity, namely, the capacity to generate both primary AFCs and memory B cells responsive to lethal challenge. Immunization by infection is the most efficient means of generating protective memory B cells, in contrast to subunit vaccine. The data also indicate that memory AFCs are predominantly localized to the regional lymphoid tissue where challenge HA is found, unlike primary AFCs, which are restricted to the priming site and which require in vivo CD4+ T-cell help.

Human Parainfluenza Virus Type 1 Immunization of Infant Mice Protects from Subsequent Sendai Virus Infection

Human parainfluenza virus type 1 (hPIV-1) infections are a common cause of "croup" and hospitalizations among young children, yet no vaccine is yet available. Sendai virus (mouse PIV-1) is the closest known homologue of hPIV-1. Here we address the possibility of using a xenotropic, nonpathogenic PIV as a vaccine in infants, by assessing the efficacy of hPIV-1 vaccination of infant mice against a subsequent challenge with Sendai virus. hPIV-1 was administered intranasally to mice age 3-6 days and shown by serum antibody ELISA and elispot analysis to elicit virus-specific IgM and isotype-switched antibody-forming cells (AFC). The response was completely cross-reactive between hPIV-1 and Sendai virus. Mice were challenged with Sendai virus 6-8 weeks later and generated AFC and serum antibody responses composed of IgM, as well as IgG and IgA, unlike challenged, age-matched controls. The high IgM response among AFC was not seen in mice primed as adults with hPIV-1 and challenged with Sendai virus. The hPIV-1 priming of infant mice afforded protection, as the majority of these mice survived the lethal Sendai virus challenge, as did all adult primed animals. These data support the notion that the unmodified xenotropic Sendai virus might function effectively in human infants as a vaccine against hPIV-1.

Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective.

This study investigated whether a single amino acid change in the hemagglutinin (HA) molecule influenced the efficacy of formalin-inactivated influenza A (H3N1) vaccine candidates derived from high-growth reassortants between the standard donor of high-yield genes (A/PR/8/34 [H1N1]) and host cell variants generated from the same clinical isolate (A/Memphis/7/90 [H3N2]) by passage in embryonated chicken eggs. Two clones of the isolate generated by growth in eggs differed from the parent virus (represented by an MDCK cell-grown counterpart) solely by the presence of Lys (instead of Glu) at position 156 or Ile (instead of Ser) at position 186 in the HA1 subunit. The protective efficacy of egg-grown HA Lys-156 and HA Ile-186 reassortant variants was compared with that of the MDCK cell-grown reassortant vaccine. Classically, antibody titers in serum have been used to demonstrate vaccine efficacy. Here, parameters of B-cell responsiveness were monitored, including the kinetics, character, and localization of the primary antibody-forming cell (AFC) response and the development of B-cell memory in lymphoid tissues associated with the priming site (spleen) and responsive to pulmonary challenge with infectious virus (upper and lower respiratory tract lymph nodes). We show that the egg-grown HA Lys-156 variant induced an AFC profile vastly different from that elicited by the other two reassortant vaccines. The vaccine was poorly immunogenic; it induced antibodies that were cross-reactive prior to challenge but which, postchallenge with a lethal dose of the MDCK cell-grown reassortant virus, were targeted primarily to the HA Lys-156 variant, were of the immunoglobulin M isotype, were nonprotective, and were derived from the spleen. In contrast, the egg-grown HA Ile-186 variant was remarkably like the MDCK cell-grown virus in that protective immunoglobulin G antibodies were unaffected by the Ile-186 substitution but poorly recognized HA with Lys-156. Furthermore, memory AFC responsiveness was localized to regional lymphoid tissue in the upper respiratory tract, where challenge HA was found. Thus, it is recommended that in the selection of vaccine candidates, virus populations with the egg-adapted HA Lys-156 substitution be eliminated and that, instead, egg-grown isolates which minimally contain Ile-186 be used as logical alternatives to MDCK cell-grown viruses.

Immunotoxicologic studies with CI-959, a novel benzothiophene cell activation inhibitor

CI-959 is an orally effective inhibitor of cellular activation in both in vitro and animal models. To assess the effects of CI-959 on immune function, male Fischer 344 rats were evaluated for splenic T- and B-lymphocyte populations, antibody-forming cell response to sheep red blood cells (sRBC), concanavalin A and pokeweed mitogen-induced lymphocyte proliferation, Natural Killer cell activity, and reticuloendothelial system clearance of sRBC. Host resistance was measured in female B6C3F1 mice using Listeria monocytogenes, Streptococcus pneumonia, and B16F10 melanoma models. CI-959 was administered to both species of rodents at 25, 50, and 75 mg/kg/day for 14 days. A vehicle control and two positive controls (cyclophosphamide and dexamethasone) were run concurrently. CI-959 generally did not suppress immunological responses in rats at doses lower than those which also altered body weight gain and reduced spleen and thymus weights. Natural Killer cell activity was significantly reduced at 50 and 75 mg/kg CI-959. At 75 mg/kg rats also exhibited a reduction in ability to make anti-sRBC antibody. The number of T- and B-lymphocytes, proliferative response to mitogens, and macrophage activity of the reticuloendothelial system were not affected by CI-959. CI-959 also did not alter resistance of mice to Listeria monocytogenes, Streptococcus pneumoniae, or B16F10 melanoma cells. Based on these ex vivo and in vivo assays, the rodent immune system does not appear to be a sensitive or lexicologically important target for CI-959.

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.

Distinctive Kinetics of the Antibody-Forming Cell Response to Sendai Virus Infection of Mice in Different Anatomical Compartments

The single-cell ELISPOT assay was used to determine the frequency and isotype commitment of virus-specific antibody-forming cells (AFC) at different anatomical locations following intranasal Sendai virus infection of C57BL/6 and 129/Sv mice. AFC responses in the mediastinal and cervical lymph nodes showed sharp increases and declines, first of IgM AFC, peaking about 7 days after infection, and then of IgG and IgA AFC, peaking about 10 days after infection. A wave of IgM AFC preceding the other isotypes was less evident in the spleen, where peak frequencies of AFC occurred 14 days after infection. Virus-specific AFC appeared in the bone marrow with a unique kinetic pattern, increasing in frequency gradually over the first 3 weeks after infection to a plateau that remained constant. Circulating IgM and IgG achieved significant titers approximately a week after infection; IgM titers were transient, but IgG levels increased sharply and remained high, reflecting the longevity of the bone marrow AFC response. Strain differences in isotype bias were noted, particularly preferential switching to the γ2a gene in 129/Sv mice. The B-cell response to acute respiratory viral infection thus exhibits features that are distinct from the primary response to nonreplicating antigens.

Cyclic AMP Is an Essential Factor in Immune Responses

In the present studies, cAMP, a secondary messenger historically viewed as a negative mediator of immune responses, was demonstrated to possess immunoenhancing activity at low concentrations. In parallel experiments the adenylate cyclase inhibitor, 2′,3′-dideoxyadenosine, produced a marked inhibition of humoral and proliferative immune responses suggesting that cAMP at physiologically relavent concentrations acts as a critical second messenger in immune responses. Direct addition of dibutyryl cAMP (10 - 100 μM), a membrane permeable cAMP analog, to mouse spleen cell cultures produced a marked and dose-related increase (25-100%) in humoral immune responses as measured by the primary IgM antibody forming cell response to the antigen, sheep erythrocytes. Over a similar concentration range, dibutyryl cAMP (5 - 50 μM) also dose-dependently enhanced (25-50%) phorbol 12-myristate 13-acetate/ionomycin-stimulated lymphoproliferation. Incubation of spleen cells with 2′,3′-dideoxyadenosine (40 - 80 μM), an adenylate cyclase inhibitor, for 30 min depressed significantly the basal level of intracellular cAMP. 2′,3′-dideoxyadenosine treatment also significantly decreased both the antibody forming cell response and the proliferative response in a dose-dependent manner. Interestingly, the antibody forming cell response exhibited significantly greater sensitivity to inhibition by 2′,3′-dideoxyadenosine than the lymphoproliferative responses. The critical role for cAMP as a positive immunoregulatory signal is further supported by the fact that the immunosuppression by 2′,3′-dideoxyadenosine could be reversed completely in the antibody forming cell response to sheep erythrocytes through the addition of dibutyryl cAMP into the culture medium. Partial but not complete reversal of the inhibitory effects of 2′,3′-dideoxyadenosine on lymphoproliferation was also demonstrated by dibutyryl cAMP. Taken together, these results suggest that cAMP acts as a positive regulatory signal for immune responses as indicated by the fact that depletion of intracellular cAMP induces a marked inhibition of humoral and proliferative responses.

Modification of immune response in mice by ciprofloxacin

Some studies have suggested that the addition of ciprofloxacin to in vitro cultures of mitogen-stimulated lymphocytes exerts inhibitory effects on cell cycle progression and immunoglobulin (Ig) secretion. We tested the effects of this drug on some immunity parameters in BALB/c mice. Mice treated intraperitoneally with ciprofloxacin (10 mg/kg of body weight per day) for 3 consecutive days and immunized with sheep erythrocytes 24 h after the last injection showed significant suppression of hemolytic IgG-forming cells, whereas the response of IgM-forming cells remained unchanged. When treatment lasted 7 days the response of antibody-forming cells was not modified. When the 3-day treatment was started at 24 h after immunization with sheep erythrocytes, the response of IgM-forming cells was increased, whereas the response of IgG-forming cells was suppressed. Delayed-type hypersensitivity to sheep erythrocytes was significantly suppressed in animals that received the 3-day treatment with ciprofloxacin and were immunized subcutaneously 24 h after the last injection. In vitro proliferation of lymphocytes from ciprofloxacin-treated mice in response to either lipopolysaccharide or concanavalin A was also suppressed. Leukopenia and an increase in the level of granulocyte-macrophage colony-forming cells in bone marrow were also observed in ciprofloxacin-treated mice. These results, together with those from other reports, suggest that modification of the biological responses by ciprofloxacin is a complex phenomenon that may be influenced by several factors.

Neuropeptide-mediated regulation of hapten-specific IgE responses in mice. I. Substance P-mediated isotype-specific suppression of BPO-specific IgE antibody-forming cell responses induced in vivo and in vitro.

The ability of substance P (SP) to regulate peak benzyl-penicilloyl (BPO)-specific IgE antibody-forming cell (AFC) responses in vivo and the ability of SP and other neuropeptides to regulate BPO-specific memory IgE AFC responses induced in vitro was determined. SP injected subcutaneously into BPO-keyhole limpet hemocyanin (BPO-KLH)-sensitized mice at the time of peak IgE responses suppressed these responses within 48 h (> 90%). The suppression obtained was IgE isotype-specific, dose-dependent, and transient. When spleen cells from immunized mice were cultured for 5 days with BPO-KLH, peak memory IgE AFC responses were induced in vitro. Inclusion of either SP or vasoactive intestinal peptide (VIP), but not neurotensin, serotonin, somatostatin, or gastrin, in cultures suppressed these responses in isotype-specific, dose-dependent fashion (approximately 70%). SP-, but not VIP-mediated suppression of IgE responses was abrogated by inclusion of anti-IFN gamma culture.

Effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on interleukin-4-mediated mechanisms of immunity

Because of similarities in the independent actions of the pleiotropic cytokine, interleukin-4 (IL-4), and the environmental contaminant, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), on murine B-lymphocytes suggested in earlier studies, we have investigated whether the immunosuppression mediated by direct exposure to TCDD in vitro is due to an IL-4-like biological activity. In particular, the ability of TCDD to mimic hallmark responses of B-cells to IL-4, such as upregulation of major histocompatibility complex (MHC) antigens of the class II type, increases in cell surface expression of the low affinity form of the Fc receptor for IgE (CD23) and induction of immunoglobulin class switching, was tested. At concentrations that readily suppress B-cell proliferative and antibody-forming cell responses, TCDD failed to demonstrate any of the activities of IL-4 observed in parallel cultures. Further, in experiments in which TCDD was preincubated with B-cells before addition of IL-4, no evidence of increased IL-4 activity was observed. Rather, TCDD preincubation resulted in decreased secretion of IgGI and IgE in B-cell cultures stimulated to undergo immunoglobulin class switching by incubation with bacterial lipopolysaccharide (LPS) and IL-4. Because TCDD produced comparable suppression of IgM secretion induced by LPS alone (i.e., no IL-4), it appears that TCDD inhibits the formation of fully differentiated B-cells capable of secreting antibody and has no effects on class switching events per se. Coupled with previous reports from this and other laboratories, these observations indicate that TCDD is able to suppress secretion of several classes of immunoglobulin.

Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism

Cannabinoid compounds, including the major psychoative component of marihuana, Δ 9-tetrahydrocannabinol ( Δ 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that Δ 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a G 1-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of Δ 9-THC (22 μM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 μM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and the sheep erthrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10–100 μM). Inhibition of the sRBC AFC response by both Δ 9-THC (22 μM) and CP-55940 (5.2 μM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1–100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation of intracellular cAMP. These results indicate that inhibition of the sRBC AFC response by cannabinoids is mediated, at least in part, by inhibition of adenylate cyclase through a pertussis toxin-sensitive G i-protein coupled cannabinoid receptor. Additionally, these studies further support the premise that cAMP is an important mediator of lymphocyte activation.

Immune response following oral administration of cholera toxin B subunit to HIV-1-infected UK and Kenyan subjects

To determine the effect of HIV-1 infection on immunoglobulin (Ig) G and IgA antibody response and circulating antibody forming cell response to oral immunization with the B subunit of cholera toxin. Healthy UK volunteers, and HIV-1-positive UK and Kenyan volunteers at different clinical stages of HIV-1 infection received two oral immunizations. CD4+ T cells, serum beta 2-microglobulin and neopterin were measured as surrogate markers of disease stage, and correlated with immunization response. Serum antitoxin IgG and IgA measured by enzyme-linked immunosorbent assay and antitoxin IgG, IgA and IgM antibody-forming cells detected by enzyme-linked immunospot assay at different times after two oral immunizations. UK HIV-positive volunteers (mean CD4+ T cell count, 52 x 10(6)/l) responded poorly to primary and booster immunization. HIV-infected Kenyans (752 x 10(6)/l CD4+ T cells) had a significant primary and booster antibody response, whereas those with a mean CD4+ T cell count 186 x 10(6)/l had an insignificant primary, but significant booster response. Two oral immunizations induced antibody responses in HIV-positive Kenyan groups (who may have prior immunity from exposure to environmental bacterial toxins) of similar or greater magnitude to healthy UK volunteers. Mucosal immunization may recall immune memory and be of benefit in early and moderately advanced clinical HIV disease. The findings have important clinical implications in that mucosally targeted vaccines are potentially useful in this group of patients.