Trinitrophenyl-lipopolysaccharide Research Articles

Regulation of thymus-dependent and thymus-independent production of immunoglobulin G subclasses by Galpha<sub>12</sub> and Galpha<sub>13</sub>

BackgroundA previous study from this laboratory showed that Gα12 members participate in the production of inflammatory cytokines. In spite of the identification of B cell homeostasis responses regulated by Gα13, the functional roles of Gα12 members in the production of immunoglobulin (Ig) isotypes remained unknown. This study investigated whether Gα12 members are involved in the Ig isotype antibody production with the purpose of establishing their functions in thymus-dependent and thymus-independent humoral responses.ResultsMice lacking Gα12 and/or Gα13 showed an impaired antigen-specific antibody production promoted by challenge(s) of ovalbumin or trinitrophenyl-lipopolysaccharide (TNP-LPS), used for thymus-dependent and thymus-independent stimuli, respectively. Homozygous knockout (KO) of Gα12 or double heterozygous KO of Gα12/Gα13 significantly reduced the antigen-specific total IgG level after multiple ovalbumin immunizations with decreases in the production of IgG1, IgG2a and IgG2b subclasses, as compared to wild type control. In contrast, IgM production was not decreased. Moreover, mice deficient in Gα12 or partially deficient in Gα13 or Gα12/Gα13 showed significantly low production of IgG2b in response to TNP-LPS. In TNP-LPS-injected mice, IgG1 and IgG2a productions were unaffected by the G protein KOs.ConclusionOur results demonstrate that both Gα12 and Gα13 are essentially involved in thymus-dependent and independent production of IgG subclasses, implying that the G-proteins contribute to the process of antigen-specific IgG antibody production.

Multiple acute temperature stress affects leucocyte populations and antibody responses in common carp, Cyprinus carpio L.

Stress is a potential factor causing increased susceptibility of fish to pathogens. In this study, stress-induced immunological changes that may contribute to a decreased immune status were investigated. A 3 h drop in ambient water temperature of 9 °C was used as a relative mild and acute stress model for carp. Effects of this stressor on the dynamics of leucocyte populations were determined with specific monoclonal antibodies. The relative number of circulating B-lymphocytes in the total leucocyte population decreased significantly within 4 h after the onset of single or multiple cold shocks. This decrease was reversible, as B-lymphocyte numbers were restored within 24 h. Most probably, a redistribution of B-lymphocytes contributed to this phenomenon. In head kidney, an increase was measured in the relative number of B-lymphocytes. Granulocyte numbers showed opposite reactions: the percentage of granulocytes in the total leucocyte population nearly doubled in circulation and decreased significantly in the head kidney. This demonstrates that in vivo, a mild stressor differentially alters the distribution of leucocytes. In stressed carp, the percentage of apoptotic lymphocytes in blood is significantly higher compared with the unstressed animals. B-lymphocytes as well as Ig −lymphoid cells contributed to this increased apoptosis. Labelling of blood lymphocytes with a polyclonal antiserum against the glucocorticoid receptor also showed, besides B-lymphocytes, part of the Ig −lymphoid cell population to be glucocorticoid receptor positive. As the distribution of B-lymphocytes was substantially affected, the effect of temperature stress on T-lymphocyte-independent (trinitrophenyl–lipopolysaccharide) and T-lymphocyte-dependent (dinitrophenyl–keyhole limpet hemocyanin) humoral antibody responses was determined. Kinetics of the primary antibody response to the T-lymphocyte-independent antigen showed lower antibody titres in stressed carp during the onset of the immune response, implying a slower development of the antibody response against the T-lymphocyte-independent antigen.

Comparison of the T Cell-Independent Antibody Response of Mice and Rats Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces adverse effects on the immune system of experimental animals. In this study, the effect that TCDD has on the antibody plaque-forming cell (PFC) response to the T cell-independent (TI) antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) was compared in adult female B6C3F1 mice and F344 rats. Mice or rats were given a single intraperitoneal injection of TCDD at doses ranging from 1 to 30 μg/kg, 7 days prior to immunization with TNP-LPS by intravenous injection. Three days later body, spleen, thymus, and liver weights were measured and the PFC response to TNP-LPS was determined. Thymus weights were decreased at 10 and 30 μg TCDD/kg, whereas spleen weights were decreased and liver weights increased in mice dosed at 3, 10, and 30 μg/kg. Mice dosed at 10 and 30 μg TCDD/kg had suppressed PFC responses and serum hemagglutination titers. In rats, thymus weights were decreased and liver weights increased at 3, 10, and 30 μg TCDD/kg; however, the PFC response and serum hemagglutination titers to TNP-LPS were suppressed only at 30 μg/kg TCDD. TCDD did not affect splenic lymphocyte subsets evaluated by flow cytometry. These results indicate that TCDD suppresses the TI antibody response to TNP-LPS in both B6C3F1 mice and F344 rats, with mice more sensitive to suppression by TCDD than rats.

Repeated high dose oral exposure or continuous subcutaneous infusion of 2-methoxyacetic acid does not suppress humoral immunity in the mouse

2-Methoxyethanol (ME) has been shown to be immunosuppressive in rats but not mice, with oxidation of ME to 2-methoxyacetic acid (MAA) being a prerequisite for immunosuppression. MAA is more rapidly cleared by mice than rats, consequently this study was designed to determine if increasing the bioavailability of MAA in mice might play a role in this species difference. Female B6C3F1 mice were given MAA by oral multiple daily high doses or by continuous subcutaneous infusion via mini-osmotic pumps. Humoral immunity was evaluated in MAA-exposed mice using the plaque-forming cell (PFC) response to either sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). Female F344 rats were also used to compare the effects of multiple daily MAA exposure on these humoral immune responses. Rats and mice were dosed orally twice a day for 4 days by gavage with MAA at dosages ranging from 40-320 mg/kg/day and 240-1920 mg/kg/day, respectively. All animals were immunized on the first day of dosing and body and lymphoid organ weights and PFC responses to SRBC or TNP-LPS were evaluated 4 days later. While body weights in rats were unaffected, thymus weights were reduced at all dosages of MAA and spleen weights were reduced at 160 or 320 mg/kg/day. PFC responses to SRBC and TNP-LPS were suppressed in rats at dosages of 160 and 320 mg/kg/day. In contrast, thymus weights of mice were reduced only at 960 mg/kg/day or greater, with no effect on spleen or body weights. Furthermore, neither the PFC response to SRBC nor the response to TNP-LPS was suppressed in mice exposed to any oral dosage of MAA. In the continuous infusion study, mice were subcutaneously implanted with mini-osmotic pumps containing MAA which was delivered at 840 mg/kg/day over a 7-day period. Continuous exposure to MAA via mini-osmotic pumps did not suppress the PFC response to either SRBC or TNPLPS, but rather significantly enhanced the response to TNP-LPS. These results indicate that mice are insensitive to MAA even at the high dosages given as a bolus or continuously over 1 week. The data further support earlier work, which suggested that the observed difference between rats and mice for MAA-induced immunosuppression appears to be unrelated to the availability of MAA to target lymphoid tissue in these rodent species.

Comparison of the T Cell-Independent Antibody Response of Mice and Rats Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces adverse effects on the immune system of experimental animals. In this study, the effect that TCDD has on the antibody plaque-forming cell (PFC) response to the T cell-independent (TI) antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) was compared in adult female B6C3F1 mice and F344 rats. Mice or rats were given a single intraperitoneal injection of TCDD at doses ranging from 1 to 30 micrograms/kg, 7 days prior to immunization with TNP-LPS by intravenous injection. Three days later body, spleen, thymus, and liver weights were measured and the PFC response to TNP-LPS was determined. Thymus weights were decreased at 10 and 30 micrograms TCDD/kg, whereas spleen weights were decreased and liver weights increased in mice dosed at 3, 10, and 30 micrograms/kg. Mice dosed at 10 and 30 micrograms TCDD/kg had suppressed PFC responses and serum hemagglutination titers. In rats, thymus weights were decreased and liver weights increased at 3, 10, and 30 micrograms TCDD/kg; however, the PFC response and serum hemagglutination titers to TNP-LPS were suppressed only at 30 micrograms/kg TCDD. TCDD did not affect splenic lymphocyte subsets evaluated by flow cytometry. These results indicate that TCDD suppresses the TI antibody response to TNP-LPS in both B6C3F1 mice and F344 rats, with mice more sensitive to suppression by TCDD than rats.

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

Investigations into the ubiquitous nature of high or low immune responsiveness after divergent selection for antibody production in common carp ( Cyprinus carpio L.)

This paper reports on the selection of individual carp with a high or low antibody response, in combination with reproduction by gynogenesis, in order to develop well-characterised inbred carp lines consisting of practically unlimited numbers of carp with the same genotype. Two homozygous progenies, previously characterised as having a high or low immune response to dinitrophenyl keyhole limpet haemocyanin (DNP-KLH), were immunised with either a T-dependent (DNP-human serum albumin (DNP-HSA)) or T-independent (trinitrophenyl lipopolysaccharide (TNP-LPS)) hapten-carrier complex. In comparison with the antibody response after DNP-KLH immunisation, the response to DNP-HSA was observed to be highly variable and did not differ between the divergently selected progenies. This suggests that the divergent selection for antibody production to DNP-KLH has been carrier-specific. Immunisation with T-independent TNP-LPS induced a very rapid response which differed between the high and low responders, and likely measured changes in the DNP-specific precursor pool of B cells caused by the selection. A number of selected individuals with a high immune response to DNP-KLH were infected with Trypanoplasma borreli, a haemoflagellate parasite of carp, to examine a possible relationship between the increase in immune responsiveness and disease resistance, but no change could be detected. However, individual homozygous carp were able to escape inbreeding depression and survive the infection. Such carp would likely candidates for gynogenetic reproduction to obtain viable inbred carp lines.

Immunological effects of 2-methoxyethanol administered dermally or orally to Fischer 344 rats

Exposure of rats to 2-methoxyethanol (ME) by gavage for 10 consecutive days results in immunotoxicity. To determine whether dermal exposure to ME also induces immunotoxicity, undiluted ME was applied to Fisher 344 male rats at dose levels of 150, 300, 600, 900 or 1200 mg/kg/day on shaved occluded test sites for 4 consecutive days. Decreased thymus weights were produced by all doses of ME, while reductions in spleen weight were observed at doses of 900 mg/kg/day ME or greater. The alterations in these lymphoid organ weights were produced in the absence of loss in body weight. The lymphoproliferative (LP) responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were enhanced at 1200 mg/kg/day ME compared with water controls. Separate groups of rats, employed for the antibody plaque-forming cell (PFC) response to either trinitrophenyl-lipopolysaccharide (TNP-LPS) or sheep red blood cells (SRBC), were exposed dermally to 150, 300 or 600 mg/kg/day ME for 4 consecutive days. A reduction in the PFC response to TNP was observed at 600 mg/kg/day ME, whereas decreases in the PFC response to SRBC were observed at dosages of 300 and 600 mg/kg/day ME. To compare the immunotoxic effects of dermally applied ME to those effects caused by ME administered orally, rats were dosed by gavage with 25, 50, 100 or 200 mg/kg/day ME in distilled water for 4 consecutive days. Reductions in thymus weights were observed at oral dosages ranging from 50–200 mg/kg/day, while spleen weights were reduced in rats dosed at 200 mg/kg/day ME. LP responses to PHA, PWM and Salmonella typhimurium were increased at the 200 mg/kg/day ME dose level. PFC responses to TNP-LPS and SRBC were suppressed at the 50, 100 and 200 mg/kg/day ME dosages. These results indicate that, like oral exposure, dermal exposure to ME compromises the ability of the immune system to mount an effective humoral immune response.

An enzyme-linked immunosorbant assay (ELISA) specific for antibodies to TNP-LPS detects alterations in serum immunoglobulins and isotype switching in C57BL/6 and DBA/2 mice exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin and related compounds

An enzyme-linked immunosorbant assay (ELISA) was developed to detect IgM and IgG antibodies specific for trinitrophenyl-lipopolysaccharide (TNP-LPS). Treatment of C57BL/6 and DBA/2 mice with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and other aryl hydrocarbon (Ah) receptor agonists followed by immunization with TNP-LPS resulted in a dose-dependent decrease in serum IgM which paralleled the decrease in the splenic PFC response. The ED 50 values for the IgM and splenic PFCs in C57BL/6 mice for 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (pentaCB) and 3,3′,4,4′,5,5′-hexaCB were 2.8 and 1.6, 11 and 14, and 25 and 20 μg/kg, respectively; in the less Ah-responsive DBA/2 mice, the ED 50 values were 8.5 and 10, 61 and 69, and 73 and 71 μg/kg, respectively. In addition, treatment of C57BL/6 mice with TCDD resulted in alterations of serum IgG relative to IgM and a delay of isotype switching was observed after immunization and boosting with TNP-LPS. This ELISA may prove to be a useful tool in monitoring immune function during long-term exposure of mice to TCDD and related compounds and exploring the mechanism of Ah receptor-mediated immunosuppression.

Species and strain comparisons of immunosuppression by 2-methoxyethanol and 2-methoxyacetic acid

2-Methoxyethanol (ME) and its principal metabolite 2-methoxyacetic acid (MAA) have been shown in our laboratory to be immunosuppressive in male Fischer 344 rats. In this study several strains of 12-week-old female rats and mice were used to compare the immunosuppressive activity of equimolar concentrations of ME and MAA on the trinitrophenyl-lipopolysaccharide (TNP-LPS) antibody plaque-forming cell (PFC) response, which we previously demonstrated to be a sensitive end point. Female inbred Lewis, Fischer 344 and Wistar/Furth, and outbred Sprague-Dawley rats were dosed by gavage with either ME or MAA at dosages of 0.33 to 2.64 mmol/kg/day for 10 consecutive days. Female inbred C3H and C57BL/6J, hybrid B6C3F 1, and outbred CD-1 mice were similarly dosed with equimolar dosages of 0.66 to 5.28 mmol/kg/day ME or MAA. All animals were immunized on day 9 of dosing and PFC responses evaluated 3 days later. Suppression of the PFC response was observed in all strains of rats at 2.64 mmol/kg/day ME or MAA. Lewis and Wistar/Furth rats were found to be the most sensitive strains with suppression at levels as low as 0.66 mmol/kg/day ME or MAA. While ME and MAA dosing resulted in suppression of the TNP PFC response in all the rat strains tested, such treatment did not suppress this PFC response in any of the mouse strains examined. These results indicate that under the conditions of this study rats, but not mice, are immunosuppressed by ME and MAA exposure, and that the susceptibility to immunosuppression differs among rat strains.

Enteric coated microspheres as an oral method for antigen delivery to salmonids

A comparative study was conducted to determine the immunisation efficacy of enteric coated antigen microspheres (ECAMs) versus intraperitoneal (i.p.) and immersion antigen delivery. Anal intubation of antigen served as a positive control by directly delivering the antigen to the hindgut. Naive juvenile coho salmon Oncorhynchus kisutch were exposed to three different concentrations of trinitrophenylated lipopolysaccharide (TNP-LPS) or trinitrophenylated keyhole limpet haemocyanin (TNP-KLH) by the three routes, oral (ECAM), intraperitoneal, immersion or anal intubation. TNP-LPS or TNP-KLH were prepared for oral immunisation by coating onto 0·45 mm dextrose sugar beads followed by an application of Eurdragit LD-30 co-polymer. Serum was taken from immunised and non-immunised control fish fed diet containing nonantigen coated beads. The serum was assayed by ELISA at 4, 6 and 8 weeks post-immunisation to assess anti-TNP antibody titres. Statistical analysis of the serum showed that oral vaccination significantly increased anti-TNP titres at 4 and 6 weeks post-vaccination as compared with the controls (P<0·05). The anti-TNP titres after TNP-LPS i.p. immunisation were significantly higher than the controls (P<0·05) at 4, 6 and 8 weeks post immunisation, while the group that was immersed showed no significant difference from the control. There was no significant difference between TNP-LPS i.p. and ECAM immunisation methods.The TNP-KLH immunised ECAM group showed significantly elevated levels of anti-TNP antibodies compared with controls at 6 and 8 weeks, while the i.p. immunised group was significantly higher than the control throughout the 8-week period. The group immunised by anal intubation had significantly higher titres than controls at 6 and 8 weeks but not at 4 weeks.

In vivo cortisol administration suppresses the in vitro primary immune response of winter flounder lymphocytes

Prolonged (24-day) administration of cortisol to adult winter flounder, Pleuronectes americanus , was found to decrease the in vitro primary immune response of splenic lymphocytes as measured by the passive haemolytic plaque assay. Intraperitoneal implantation of fish with cortisol mixed into a vegetable shortening and cocoa butter vehicle resulted in elevated serum cortisol levels that were within physiological ranges. Existing techniques for piscine lymphocyte culture were modified for use with winter flounder. Longer durations of elevated serum cortisol in the fish produced a more marked suppression of the plaque-forming cell (PFC) response. The suppression was evident as early as 10 days postimplantation when winter flounder cultures were stimulated for 14 days with trinitrophenyl-keyhole limpet haemocyanin (TNP-KLH). Suppression of the PFC response was not apparent until 17 days postimplantation in those cultures stimulated with trinitrophenyl-lipopolysaccharide (TNP-LPS) and was less apparent when the culture period was lengthened from 9 days to 14 days. Our results indicate that the in vitro primary PFC response can be used to evaluate the presence of chronic stress in fish.

Influence of the Ah locus on the humoral immunotoxicity of 2,3,7,8-tetrachlorodibenzo- p-dioxin: Evidence for Ah-receptor-dependent and Ah-receptor-independent mechanisms of immunosuppression

There are conflicting reports in the literature regarding the role of the Ah locus in 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) immunotoxicity. The present studies have utilized two congenic strains of C57Bl 6 mice that differ only at this locus to assess its influence on TCDD-induced suppression of antibody responses. Mice were given a single oral dose of TCDD 2 days prior to challenge with sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). The subsequent dose-dependent effects of TCDD on the amount of antibody produced by splenic plasma cells were measured using the hemolytic antibody isotope release assay. In addition, the relative importance of the Ah genotype of lymphoid versus nonlymphoid tissue was examined in adoptive transfer experiments. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced in Ah bb mice by a dose of 0.5 μg/kg TCDD and maximally induced by a dose of 2 μg/kg. Ah dd mice required 10-fold higher doses of TCDD to induce comparable levels of AHH. The degree of thymic involution and liver hypertrophy induced by TCDD was also influenced by the Ah genotype of the animals. Both Ah bb and Ah dd mice exhibited dose-dependent suppression of the anti-TNP response following TCDD exposure. The ID50 was 7.0 μg/kg in Ah bb mice and 30.8 μg/kg in Ah dd mice. Suppression of the antibody response to SRBC was also dependent on the Ah locus. The ID50 in Ah bb mice was 0.6 μg/kg TCDD. However, an apparent biphasic dose response for suppression of the anti-SRBC response in Ah dd mice suggested the involvement of an Ah-independent component of suppression as well. In adoptive transfer studies, lymphocytes were identified as an Ah-dependent component of the response. The Ah-independent component of the response was not identified, and could be either lymphoid or nonlymphoid in nature. The possibility that T helper cells represent the Ah-independent component is discussed.

Primary antibody responses to thymus-independent antigens in the lungs and hilar lymph nodes of mice

B lymphocytes from the pulmonary lymphoid tissues were stimulated with a variety of thymus-independent (TI) antigens by intratracheal (i.t.) immunization. Immune responses in the lungs and hilar lymph nodes (HLN), which are part of the localized lymphoid tissue, as well as in the spleen, the systemic lymphoid organ, were studied. Thus, primary i.t. immunization of mice with the TI-1 antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) elicited both antigen-specific and polyclonal plaque-forming cell responses from HLN, lung, and splenic B lymphocytes. These responses appeared as early as 3 days after immunization and declined by day 7. Similar immunization with another TI-1 antigen, TNP-Brucella abortus, resulted in anti-TNP responses in both pulmonary and systemic lymphoid tissues, although the kinetics of the antibody response were different than those to TNP-LPS. Interestingly an i.t. immunization with a TI-2 antigen, TNP-Ficoll, failed to induce an anti-TNP PFC response from HLN and lung B cells, although there was good antibody formation from splenic B cells. Antibody response to TNP-Ficoll was restored in pulmonary tissues when mice were immunized with TNP-Ficoll mixed with unconjugated B. abortus. In conclusion, our results indicate that TI-1 and TI-2 antigens differ in their ability to induce antibody responses in the pulmonary lymphoid tissues. The inability of TNP-Ficoll to elicit an antibody response in pulmonary lymphoid tissues has significance in the development of vaccines containing bacterial polysaccharides.

The in situ Immune Response in Popliteal Lymph Nodes of Mice After Macrophage Depletion. Differential Effects of Macrophages on Thymus-dependent and Thymus-independent Immune Responses

Mice were subcutaneously (SC) injected in the left hind footpad with dichloromethylene diphosphonate (C1 2MDP)-containing liposomes to eliminate macrophages lining the subcapsular sinus (SCS) and those in the medulla of draining popliteal lymph nodes (PLN). In order to study the effect of depletion of these macrophages on the in situ immune response in the PLN, liposome-treated mice were SC injected in the same footpad with thymus-independent (TI) type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNPFicoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH). No major differences were observed in antibody-serum titers of liposome-treated and control animals. After primary as well as secondary immunization with the TD-antigen TNPKLH, an increase in the number of antibody-forming cells (AFC) was found and the peak of response was delayed in the PLN of liposome-treated animals. Such differences were not observed with the TI-antigens. These results indicate that macrophages lining the SCS and those in the medulla of the PLN are not essential for the induction of an immune response. The positive effect of macrophage-depletion on the number of AFC may be explained by competition for the antigen by macrophages and other antigen-presenting cells.

Identification of t-helper cells as the target of stearic acid-inhibition in primary antibody responses in vitro

We have previously shown that albumin-complexed stearic acid (18:0) inhibited in vitro primary anti-TNP plaque-forming cell (PFC) responses to trinitrophenyl keyhole limpet hemocyanin (TNP-KLH), but did not affect primary PFC responses to trinitrophenyl lipopolysaccharide (TNP-LPS). The present studies were done to identify the cellular target of fatty acid inhibition. The addition of 18:0 at the initiation of antibody cultures exerted a dose-dependent inhibitory effect on subsequent PFC responses to TNP-KLH, and removal of the fatty acid after 20 h did not reverse its inhibitory effect. Preincubation of isolated T-cells with TNP-KLH and 18:0 resulted in a similar inhibition of subsequent PFC responses, but a preincubation of isolated B-cells had no effect. The addition o of 18:0 to the culture system in vitro led to a marked reduction in the level of IL-2 detectable in culture supernatants, and PFC responses could be restored by providing exogenous mouse recombinant IL-2. The addition of antigen-primed T-helper cells to antibody cultures partially abrogated the inhibition by 150 μM 18:0, apparently due to their greater production of IL-2. Lastly, following overnight incubation of unfractionated splenic lymphocytes in the presence of TNP-KLH and [1- 14C]-18:0, B-cells were shown to contain nearly 5-fold more radiolabeled oleic acid (18:1) than T-cells. Collectively, these findings implicate T-helper cells as the principle target of 18:0-inhibition of primary antibody responses in vitro, possibly as a result of the inability of T-helper cells to avoid an over accumulation of stearic acid in their membrane phospholipids.

Primary in situ immune response in popliteal lymph nodes and spleen of mice after subcutaneous immunization with thymus-dependent or thymus-independent (type 1 and 2) antigens.

Mice were immunized subcutaneously with thymus-independent (TI)-type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH) in order to study the primary in situ immune response in popliteal lymph nodes (PLN) and spleen. The spleen responded more rapidly in developing specific antibody-forming cells (AFC) than the lymph nodes did, in spite of the fact that antigens reach the spleen only after passing several lymph node stations. This difference between lymph nodes and spleen in developing AFC was particularly significant with respect to the responses to TI (both type 1 and type 2) antigens. No differences in the distribution of specific AFC in PLN and spleen were observed after immunization with TI and TD antigens. Results are discussed with respect to the relative contributions of lymph nodes and spleen to immune responses to antigens injected subcutaneously.

Secondary antibody responses to thymus-independent antigens. Decline and life-span of memory.

Immunological memory has been defined by the finding that upon a secondary injection of an antigen into an animal the immune response obtained differs from the response produced after the first inoculation of the antigen, independent of the length of time that can elapse between the first and second contact with antigen. In this report we have investigated the life-span of memory to a thymus-independent antigen, trinitrophenylated lipopolysaccharide (TNP-LPS), using a cell transfer system that allows the study of the function of isolated LPS-reactive "memory" B cells from C57BL/6 mice in histocompatible LPS-nonresponder C57BL/10ScCr hosts. We found that the longer the elapse of time between the transfer of TNP-LPS-primed C57BL/6 cells and the challenge of hosts with TNP-LPS, the lower the anti-TNP serum antibody level of the secondary response, i.e. in the absence of antigen, TNP-LPS memory cells have a short life-expectancy in the adoptive hosts as they do not persist for more than one or two weeks after transfer. Our present results suggest that induction and long-term persistence of memory to TNP-LPS in adoptive hosts cannot be solely explained by the long life-span of a subpopulation of antigen-specific memory B cells, but rather through the continuous recruitment of newly formed cells and probably antigen persistence.

The immunological profile of a new immunomodulatory agent, oxamisole

Oxamisole is a T-cell immunorestorative agent when administered by the oral (p.o.) route. It has little or no effect on the IgM or IgG responses to the T-dependent antigen, sheep erythrocytes (SRBC), in normal mice but augments antibody production in immunodeficient animals. Unlike the response to SRBC, the humoral immunocompetence of both normal and immunosuppressed animals sensitized with the T-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS) was unaffected by oxamisole. Oxamisole restored cellular immunocompetence, as evidenced by an increase in the in vitro proliferative response of normal murine splenocytes to T-cell mitogens, while decreasing B-cell mitogenic responses. This indicates that oxamisole may selectively restore T-cell function. However, oxamisole did not significantly modify the classical T-cell-mediated delayed hypersensitivity responses to either the protein antigen methylated bovine serum albumin or to the contact-sensitizing antigen oxazolone. When assayed in vitro, oxamisole enhanced macrophage chemotactic function but not phagocytic function, suggesting a potential stimulation of the reticuloendothelial system. In vivo studies failed to demonstrate any consistent significant activation of murine macrophage function following oral dosing with oxamisole.

The early postnatal development of the primary immune response in rat popliteal lymph node, stimulated with thymus-independent type-1 and type-2 antigens

To examine the development of the postnatal immune response to thymus-independent type-1 (TI-type 1) and TI type-2 antigens, respectively, trinitrophenyl-lipopolysaccharide (TNP-LPS) or TNP-Ficoll was injected subcutaneously into the hind footpads of young rats of various ages. After 5 days the popliteal lymph nodes (PLNs) were removed and the localization pattern of specific anti-TNP antibody-containing cells was studied. The first specific antibody-containing cells elicited in rats by TNP-LPS appeared in animals at day 19 after birth. The results suggest that the development of these cells from lymphocyte to plasma cell occurs while they migrate from cortex to medulla. An unexpected finding was the low response to TNP-Ficoll in PLN; from 6 weeks after birth only very few specific antibody-containing cells were found. However, in the spleen numerous anti-TNP antibody-containing cells were found in the periarteriolar lymphocyte sheaths. To test the exclusive role of the spleen in the appearance of anti-TNP antibody-containing cells in lymph node after subcutaneous administration of TNP-Ficoll, the experiment was repeated in rats that had been splenectomized. Evidence from these experiments suggests that the spleen plays a major role in the appearance of the above-mentioned cells in lymph nodes.