Increased translocation of Escherichia coli and development of arthritis in vitamin A-deficient rats.

We studied the immune response and the colonization pattern in vitamin A-deficient rats that were colonized with the Escherichia coli O6 K13 pOmp 21 strain, which is genetically manipulated to produce ovalbumin and to be resistant to ampicillin. In the vitamin A-deficient rats, the number of bacteria per gram of feces was about five times higher than in the paired fed control rats 4 weeks after colonization. In the control rats, the colon and the lower part of the ileum were colonized, while in the vitamin A-deficient rats all parts of the small intestine, as well as the colon, were heavily inhabited by bacteria. Furthermore, in 75% of the vitamin A-deficient rats, the E. coli bacteria were found in the mesenteric lymph nodes, and in 50% of the rats E. coli were found in the kidneys. These animals also developed severe arthritis. The levels of serum immunoglobulin G (IgG), IgM, IgE, and biliary IgA antibodies against the bacterial antigens were significantly higher in the vitamin A-deficient rats than in the control rats. The number of IgA-producing cells in the lamina propria of the small intestine was significantly lower in the vitamin A-deficient rats than in the control rats; however, there was an increase in the number of CD8+ cells and transforming growth factor beta-producing cells in the lamina propria of the vitamin A-deficient rats. Disturbances in T-cell function were demonstrated, since spleen cells from the vitamin A-deficient rats produced more gamma interferon and interleukin-2 in vitro than control spleen cells. In summary, vitamin A deficiency led to a decrease in the ability to control the localization of intestinal bacteria and an increase in translocation, which was followed by development of arthritis regardless of substantial levels of antibacterial antibodies. The bacterial invasion made the animals hyperresponsive to the bacterial antigens, despite the fact that vitamin A deficiency is normally associated with suppressed antibody production, as previously shown by us and others.     

Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine.

To investigate the importance of vitamin A in the ability to respond to oral antigen administration, rats were fed a vitamin A-free diet. The animals were immunized perorally three times with a mixture of cholera toxin (CT) and a commercial cholera vaccine. The total immunoglobulin A (IgA) concentration as well as the specific IgA anti-CT antibody levels in serum and bile was significantly lower in the vitamin A-deficient animals than in the paired fed controls (animals that were fed a normal commercial diet in an amount equal to the amount the deficient animals consumed), while the levels of total and specific anti-CT IgG were not affected to the same extent by the vitamin A deficiency. The number of IgA anti-CT antibody-producing cells in the mesenteric lymph nodes after immunization was also significantly lower in the vitamin A-deficient rats than in the control rats. Supplementation of the diet with retinyl palmitate restored the ability to mount an IgA antibody response to the antigen, since the level of specific IgA anti-CT antibodies in relation to the total IgA concentration was as high in the vitamin A-supplemented group as in the paired fed control group. Restricted diet intake by itself did not affect the ability to respond adequately to the antigen since there was no difference in IgA anti-CT antibody level between paired fed rats and those being fed ad libitum. Assessment of transforming growth factor beta in cell cultures revealed no difference between vitamin A-deficient and paired fed animals. In summary, vitamin A deficiency resulted in a decreased number of IgA-producing cells, decreased IgA production, and a reduced ability to respond with IgA antibodies to the oral cholera vaccine.     

Functional and phenotypic analysis of human T-cell clones which stimulate IgE production in vitro.

Peripheral blood mononuclear cells (PBMC) from a patient suffering from the hyper IgE syndrome were used to generate phytohaemagglutinin (PHA)-expanded T-cell clones (all CD4+, CD8-, CD23-). A selection of the clones was tested for their ability to help IgE secretion by culturing with normal B cells in the presence of solid-phase antibody to CD3. Supernatants were harvested on Day 7 and assayed by ELISA for IgE, IgG and IgM. Lymphokine secretion by the clones was assessed by culturing clones for 24 hr with solid-phase antibody to CD3 followed by assay of the supernatants for IL-2, IL-4 and interferon-gamma (IFN-gamma) production. In addition, clones were analysed by flow cytometry for CDw29 and CD45R expression. Initial experiments with seven clones indicated that those clones that could help IgE secretion also stimulated optimal IgG and IgM responses. All clones appeared to secrete IL-2, IL-4 and IFN-gamma, although the amounts of each varied. These results confirm recent findings that human T-cell clones do not fall into Tinf (Th1) and Th (Th2) type subsets as described in the mouse. There was no clear correlation between the lymphokines secreted by the clones and their capacity to help IgE production. However, the helper function of the clones for all isotypes, including IgE, appeared to be related to the level of expression of the surface antigen CDw29.     

Inhibition of inducible nitric oxide synthase expression by interleukin-4 and interleukin-13 in human lung epithelial cells.

Oral feeding of proteins causes peripheral T-cell tolerance, as revealed by reduced delayed-type hypersensitivity (DTH) reactivity after immunization. This type of tolerance can be due both to passive T-cell anergy and active immunosuppression. Using ovalbumin-fed mice we studied whether putatively immunostimulatory cytokines could break this state of mucosal tolerance. Cytokines were administered locally at the site of attempted sensitization. It was found that neither interleukin-2 (IL-2), interferon-gamma (IFN-gamma) nor granulocyte-macrophage colony-stimulating factor (GM-CSF) could restore the response to immunization. In contrast, local administration of IL-12 at the site of attempted immunization resulted in full recovery of DTH reactivity. The dichotomy between the two Th1 stimulatory cytokines IFN-gamma and IL-12 was also reflected by different effects on ovalbumin-specific antibody isotypes. Although both IFN-gamma and IL-12 downregulated serum IgG1-levels in tolerant mice, suggesting decreased ovalbumin-specific Th2 function, only local administration of IL-12 led to increased serum IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.     

Regulation of DTH and IgE responses by IL-4 and IFN-gamma in immunized mice given pertussis toxin.

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) are cytokines with important functions in regulating immune responses. IFN-gamma may be produced by cells responsible for delayed-type hypersensitivity (DTH), whereas IL-4 is essential for IgE production. Pertussis toxin (PT) from Bordetella pertussis enhances both DTH and IgE responses, and causes enhancement of both IFN-gamma and IL-4 secretion in immunized mice. In the present study, the effects of neutralizing monoclonal antibodies against IFN-gamma or IL-4 on DTH, serum IgE and cytokine production were assessed. Treatment with a monoclonal anti-IL-4 antibody at the time of immunization caused a striking increase in DTH responses, and elicited enhanced IFN-gamma expression, while inhibition of the production of IL-4 and IgE was observed. By contrast, injection of a monoclonal anti-IFN-gamma antibody was followed by significant but not complete suppression of DTH reactions. IFN-gamma secretion was also inhibited, whereas IL-4 production and serum IgE were increased. Thus antibodies to IL-4 and IFN-gamma, given at the time of immunization, can profoundly influence the nature of short-term immune responses elicited by PT in immunized mice.     

Generation of rat Th2-like cells in vitro is interleukin-4-dependent and inhibited by interferon-gamma.

Differentiation of naive T cells into effector cells producing T helper type 1 (Th1) and Th2 cytokines is regulated by the presence of specific cytokines in the T-cell microenvironment. The effect of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) on Th1- and Th2-like cell development was investigated in cultures of mixed rat spleen cells. These cells were cultured for 4 days in medium containing concanavalin A (Con A) with or without additional IL-2, IFN-gamma or IL-4. The cells were then washed and their capacity to produce IL-4, IL-5 and IFN-gamma determined following stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Freshly isolated cells stimulated with PMA and ionomycin expressed detectable levels of IL-4 and IL-5 mRNA as measured by a quantitative polymerase chain reaction (PCR) procedure and much higher levels of IFN-gamma mRNA. Cells cultured with Con A for 4 days, washed, and restimulated with PMA + ionomycin were unable to express detectable levels of IL-4 and IL-5 mRNA, but produced high levels of IFN-gamma mRNA. Addition of IL-4, or anti-IFN-gamma antibody, to Con A-driven splenocyte cultures restored the ability of restimulated cells to express IL-4 and IL-5. CD4+ T cells isolated from these cultures also showed an increased capacity to secrete IL-4 and IL-5 when anti-IFN-gamma and IL-4 were present in the culture medium. When cultured for 4 days with Con-A, IL-4 and anti-IFN-gamma splenocytes showed an increased capacity to proliferate in response to recombinant IL-2 and proliferated in response to IL-4 alone. IL-2 had no effect on cytokine production by cultured splenocytes. These results indicate that: (1) IL-4 is essential for the generation of Th2-like cells; (2) IFN-gamma inhibits IL-4 production by mixed spleen cells and suppresses generation of IL-4 responsive T cells; (3) in mixed spleen cell cultures mitogenic stimulation favours differentiation of naive rat T cells into effector cells expressing a Th1, and not Th2, cytokine profile.     

Limiting dilution analysis of CD4 T-cell cytokine production in mice administered native versus polymerized ovalbumin: directed induction of T-helper type-1-like activation.

Polarized expression of T-helper type-1 (Th1)- or Th2-like patterns of cytokine production frequently correlates with disease outcome. Previously, we have described the long-lived reciprocal regulation of ovalbumin (OVA)-specific IgE (> 95% inhibition) and IgG2a (300-800-fold increased) production following administration of high MW OVA polymers (OVA-POL), in both de novo and ongoing OVA (alum)-induced responses. Here, limiting dilution analysis (LDA) was used to compare precursor frequencies of CD4 T cells producing interferon-gamma (IFN-gamma), interleukin-4 (IL-4) or IL-10 following OVA versus OVA-POL exposure in vivo. Adjuvants were not used, so as to circumvent their impact on measurement of precursor frequencies. We found that the two forms of antigen elicited T-cell activation of comparable intensity, as indicated by equivalent precursor frequencies of clonogenic antigen-specific CD4 T cells. However, they elicited qualitatively different cytokine responses. OVA-POL treatment led to 10-fold higher (mean of six independent LDA experiments) frequencies of IFN-gamma-producing cells, and a mean fivefold lower frequency of IL-10-producing cells, than was observed following in vivo administration of unmodified OVA. Thus, the high MW polymerized form of antigen acted to steer commitment of naive (for this antigen) CD4 T-cell activation from a situation in which IL-10 producers outnumbered IFN-gamma-producing cells by a factor of 4:1 (found in mice administered OVA), to one where IFN-gamma producers dominated by a factor of 11:1 (in mice given OVA-POL), i.e. a qualitative shift in the nature of the OVA-specific response induced from Th2-like to Th1-like. In vivo co-administration of anti-IFN-gamma monoclonal antibody (mAb) abolished the capacity of OVA-POL to preferentially elicit Th1-like dominance. Interestingly, although the ratios of IFN-gamma:IL-4 and IFN-gamma:IL-10 OVA-specific precursor frequencies were strongly increased following OVA-POL exposure (mean 18- and 47-fold higher), the frequency of IL-4-producing CD4 T cells did not differ significantly. The data suggest that this modified antigen promotes in vivo commitment of naive T cells towards a Th1-like response, with consequent inhibition of IgE and enhancement of IgG2a responses, not through direct effects on IL-4 production, but via decreased frequencies of IL-10 and increased frequencies of IFN-gamma-producing OVA-specific CD4 cells. Collectively, the data (1) demonstrate the ability to manipulate commitment of antigen-driven CD4 T-cell populations in naive mice to specific patterns of cytokine gene expression, and (2) provide in vivo evidence of the regulatory role played by IFN-gamma in limiting induction and/or expansion of IL-4- and IL-10-producing CD4 cells to protein allergens.     

Differential regulation by phorbol myristate acetate of IFN-gamma and IL-4 expression in anti-CD3 stimulated mouse spleen cells.

Expression of messenger RNA (mRNA) specific for two cytokines representative of both Th1 and Th2 cell subtypes, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) respectively, was analysed in murine spleen cells stimulated in vitro with an anti-CD3 monoclonal antibody (mAb). This stimulation induced predominantly an IFN-gamma message. In contrast, in the presence of phorbol myristate acetate (PMA), the expression of IFN-gamma was decreased whereas the IL-4 message was dramatically enhanced. These results were confirmed by Polymerase Chain Reaction (PCR) analysis and extended to include another T-cell activator [Concanavalin A (Con A)]. The results suggest the existence of a differential pathway of activation for the Th subpopulations or a differential regulation of the T helper lymphokine gene expression by PMA-induced signals.     

Hyper IgE syndrome--a disease of imbalanced activation of helper T-cell subsets?

Hyper-IgE syndrome is a rare immunodeficient disorder characterized by recurrent severe staphylococcal infections of the skin and sinopulmonary tract, chronic eczematoid rashes, coarse facial features, mild eosinophilia, and markedly elevated serum IgE levels. Hyperimmunoglobulinemia D, depressed DTH, and varying degrees of pathogenesis of this syndrome is unknown. The clinical manifestations and the recent research findings indicated the followings: 1) increased production of IL-4: hyperimmunoglobulinemia E, increased number of Fc epsilon R(+)-cells in peripheral blood, 2) defective production of IFN-gamma: abnormal local inflammatory responses (formation of cold abscesses), chemotactic defect in the circulating neutrophils (abnormalities in IFN-gamma/IL-8 pathway), depressed DTH, 3) T-cell immunodeficiency?-chronic dermatitis? 4) genetic factors (frequent familial occurrence, characteristic facial appearance with broad nasal bridge). These observations led us to postulate that both the increased production of IL-4 and the defective production of IFN-gamma may be the immunopathological bases of this syndrome. Recently, these cytokines were demonstrated to be secreted by different subsets of helper T-cells, designated TH1 and TH2, in murine system, suggesting that the regulatory imbalances between IL-4 and IFN-gamma in this syndrome might be due to the differential activation or inactivation of these helper T-cell subsets.     

Saponin adjuvant primes for a dominant interleukin-10 production to ovalbumin and to Trypanosoma cruzi antigen.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.     

The role of small intestinal antigen-presenting cells in the induction of T-cell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance.

The oral administration of soluble protein antigen results in profound immunological tolerance. However, the tissue location and function of antigen-presenting cells (APC) that stimulate this response remain unclear. We have hypothesized that the properties of cells presenting antigen to naive T cells within the gut are involved, and therefore gut APC should stimulate T-cell responses with different characteristics to those induced by other APC. To test this, we studied in vitro primary T-cell responses following presentation of soluble protein antigen by cells from the Peyer's patches (PPC) and lamina propria (LPC) of the murine small intestine and the spleen (SPLC). Each APC population stimulated antigen-specific proliferative responses with similar anamnestic characteristics; however, analysis of the cytokines produced revealed marked differences. Whereas SPLC stimulated the balanced production of T-helper type 1 (Th1) and Th2 cytokines, PPC induced a profile consistent with the provision of T-cell help for IgA production. Interestingly, presentation of antigen by LPC stimulated high levels of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) in the absence of other cytokines [interleukin-2 (IL-2), IL-4, IL-5]. Evidence from analysis of cell activation and division within the cultures suggested that this profile may result from the preferential activation of CD8+ T cells by LPC; however, the lack of conventional CD4+ T-cell cytokines indicated a defect in the normal function of these cells. Adoptive transfer of antigen-pulsed LPC to syngeneic animals abrogated the induction of delayed-type hypersensitivity (DTH) responsiveness, which followed a subsequent conventional antigen challenge further suggesting a role for lamina propria APC in tolerance induction.     

Alginic acid oligosaccharide suppresses Th2 development and IgE production by inducing IL-12 production

BACKGROUND: Since allergen-specific IgE is directly involved in the type I allergic reaction, development of a method for inhibiting Th2 responses which lead to the induction of IgE production would be a useful approach for preventing allergic disorders. The ability and mechanism of alginic acid oligosaccharide (ALGO), an oligosaccharide obtained from natural edible polysaccharide, for suppressing Th2 responses was examined in detail. METHODS: Lymph node cells obtained from beta-lactoglobulin (beta-LG)-primed BALB/c mice were cultured in vitro with an antigen for 3 days in the absence or presence of ALGO. The amount of cytokine in each culture supernatant was measured. The effect of ALGO on Th2 development was also examined by using ovalbumin specific T cell receptor transgenic mice. Antibody production in the serum of BALB/c mice that had been immunized with beta-LG or beta-LG plus ALGO was investigated. RESULTS: The production of IFN-gamma induced by antigen stimulation was upregulated by ALGO in a dose-dependent manner. IL-12 production was also enhanced by ALGO, and the addition of the anti-IL-12 antibody to the culture abrogated the effect of ALGO. On the other hand, IL-4 production by antigen-stimulated splenocytes of transgenic mice was suppressed in the presence of ALGO. Furthermore, IgE production by ALGO-treated mice was significantly inhibited compared with control mice. CONCLUSIONS: These results indicate that ALGO suppressed antigen-induced Th2 development by inducing IL-12 production. ALGO also inhibited in vivo IgE production. These findings suggest that ALGO is expected to be an edible anti-allergic agent.     

Neisserial immunoglobulin A1 protease induces specific T-cell responses in humans.

We have previously shown that immunoglobulin A1 (IgA1) protease, an exoenzyme of pathogenic neisseriae, can trigger the release of proinflammatory cytokines from human monocytic subpopulations. Here, we demonstrate a dose-dependent T-cell response to recombinant gonococcal IgA1 protease (strain MS11) in healthy human blood donors. This response was delayed in comparison to the immune response against tetanus toxoid. Stimulation with IgA1 protease led to the activation of CD4(+) and CD8(+) T cells, as well as CD19(+) B cells and CD56(+) NK cells, indicated by de novo expression of CD69. Only CD4(+) T cells proliferated and stained positive for intracellular gamma interferon (IFN-gamma). Both proliferation and IFN-gamma production were dependent on antigen presentation via major histocompatibility complex class II. Peripheral blood mononuclear cells stimulated with IgA1 protease produce IFN-gamma and tumor necrosis factor alpha but no, or very low amounts of, interleukin-10 (IL-10) or IL-4, indicating a Th1-based proinflammatory immune response. These findings support the significance of IgA1 protease as a virulence determinant of bacterial meningitis and its function as a dominant proinflammatory T-cell antigen.     

Identification of T-cell stimulatory antigens of Chlamydia trachomatis using synovial fluid-derived T-cell clones.

Intraperitoneal immunization of Hooded Lister rats with a soluble antigen such as bee venom phospholipase A2 (PLA2), or ovalbumin (OVA) together with the toxic lectin, ricin, eliminates a population of early-activated CD8+ T cells which regulate IgE production. These early-activated CD8+ T cells are eliminated because they bear increased ricin-binding glycoproteins on their surface. This immunization regimen produces a vigorous and long-lived IgE response. The effect of this treatment on the capacity of splenic T cells to secrete interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and to generate IL-4 RNA message was assessed. IFN-gamma production by phytohaemagglutinin (PHA)- or ionomycin and phorbol myristate acetate (PMA)-stimulated splenocytes or purified splenic T cells from animals immunized with antigen and ricin was substantially reduced as compared with animals which were given saline or antigen alone (P < 0.001 Student's t-test). At the height of the primary IgE response IFN-gamma production by PHA-stimulated splenocytes was positively correlated with the number of CD8+ T cells (r = 0.90, P < 0.001) and inversely related to the level of serum IgE (r = -0.77, P < 0.020); serum IgE was inversely related to the number of CD8+ T cells (r = -0.92, P < 0.001). The reduced capacity of spleen cells from ricin and antigen immunized rats to produce IFN-gamma was first seen 7 days after immunization. The fall in the ability of splenocytes to secrete IFN-gamma closely paralleled the rise in serum IgE. IL-2 was assayed using an IL-2-dependent cell line which responded to rat IL-2 but not IL-4. Production of IL-2 by splenocytes taken from rats immunized with ricin+antigen was not significantly different to that produced by comparable cells obtained from animals immunized with antigen alone or saline. However, the levels of IL-4 mRNA, detected in ionomycin and PMA-stimulated splenocytes using a quantitative polymerase chain reaction (PCR) procedure, were three- to fourfold higher in ricin and antigen immunized animals as compared with control animals. Following boosting with antigen and ricin the levels of IL-4 message detected increased a further three- to fourfold. These data show that the potentiated IgE response produced by immunization with antigen+ricin is associated with a decreased ability of splenocytes to produce IFN-gamma and an increased capacity to make IL-4.     

Defects in the regulation of anti-DNA antibody production in aged lupus-prone (NZB x NZW)F1 mice: analysis of T-cell lymphokine synthesis.

(NZB x NZW)F1 (B/W) mice spontaneously develop a lupus-like syndrome characterized by an increased level of autoantibodies in old mice. We analysed the role of T cells in the regulation of anti-DNA antibody production by B cells in vitro as a function of age. In cultures of old mouse T and B cells, IgG and IgM anti-DNA antibodies were synthesized at high levels, in contrast to consistently lower amounts, particularly of IgG, measured in cultures of young mouse cells. Addition of young mouse T cells to old B cells inhibited IgG, but not IgM, anti-DNA production, whereas T cells from old mice stimulated IgG synthesis by young mouse B cells. Addition of supernatants harvested from concanavalin A (Con A)-stimulated T cells to B-cell cultures induced similar effects. Therefore, we evaluated possible modifications of lymphokine synthesis compared to that of the healthy NZW parent. T cells from old mice were able to secrete normal levels of interferon-gamma (IFN-gamma) and interleukin (IL)-10; however, secretion of IL-2 and IL-4 was dramatically decreased. Semi-quantitative polymerase chain reaction analysis of constitutive RNA messengers showed increased IFN-gamma levels in young and old B/W mice, and normal IL-10 mRNA levels in young and higher levels in old mice. Constitutive IL-2 and IL-4 mRNA were detected only after Con A stimulation and their levels decreased in old compared to young B/W mice; in particular IL-2 mRNA was considerably lower in old B/W than in control NZW mice. Taken together, these results suggest that, despite constitutive T-cell abnormalities, young B/W mice are able partially to control their lymphokine production, whereas aged mice exhibit a deficient synthesis, associated with an increased capacity to produce IFN-gamma.     

Contact sensitization to oxazolone: involvement of both interferon-gamma and interleukin-4 in oxazolone-specific Ig and T-cell responses.

The synthesis and role of several lymphokines were examined during contact sensitization to oxazolone (OX). Application of OX to the skin of mice increased the delayed-type hypersensitivity (DTH) response to challenge, serum titres of OX-specific IgG1 and IgG2a, and draining lymph node cell (LNC) numbers. At day 3, LN contained detectable interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-2 or IL-3 mRNAs; IL-3 and higher levels of IL-4, IFN-gamma and GM-CSF mRNAs were measured after 24 hr culture with anti-CD3 antibody in OX-primed but not unprimed LNC. As a result of sensitization, LNC secreted IL-3 constitutively and produced elevated levels of IL-2, IL-3, IL-4 and IFN-gamma in response to anti-CD3 antibody; a similar but weaker lymphokine response was recalled by OX-protein conjugate. CD4+ cells were the major source of the anti-CD3-induced lymphokines except IFN-gamma, which was derived mainly from CD8+ cells. Since both IL-4 and IFN-gamma were synthesized by OX-primed LNC in vivo and in vitro, their role was investigated by administering anti-lymphokine antibodies at the time of sensitization. Anti-IL-4 treatment reduced OX-specific serum IgG1 titres without affecting IgG2a titres, whereas anti-IFN-gamma treatment reduced IgG2a but not IgG1 titres. Although neither antibody altered DTH responsiveness, anti-IFN-gamma treatment markedly increased IL-4 production by CD4+ LNC and reduced IFN-gamma production in vitro, particularly by CD4+ cells. We conclude that endogenous IL-4 and IFN-gamma reciprocally influence the isotype of the Ig response to OX and that IFN-gamma also affects the relative levels of IL-4 and IFN-gamma synthesis by CD4+ LNC.     

Role of Th1 and Th2 cells in anterior chamber-associated immune deviation.

The immunological privilege of the anterior chamber (AC) of the eye is due, at least in part, to a selective antigen-specific down-regulation of delayed-type hypersensitivity (DTH) and a normal induction of antibody responses: a phenomenon that has been termed anterior chamber-associated immune deviation (ACAID). This dichotomy in the systemic immune responses is suggestive of a T-helper type-2 (Th2)-dominated immune phenotype in which a Th2 cell population is preferentially activated and cross-regulates T-helper type-1 (Th1) effector elements. This hypothesis was tested by comparing the cytokine pattern of antigen-pulsed spleen cells from mice primed in the anterior chamber with antigens that induce ACAID with responses in hosts primed with antigens that do not induce ACAID. The results indicated that CD4+ spleen cells from hosts primed in the AC with antigens that induce ACAID produced significant quantities of interleukin-10 (IL-10) but insignificant levels of IL-2, IL-4 and interferon-gamma (IFN-gamma). In contrast, hosts primed in the AC with antigens that do not induce ACAID, but instead elicit normal DTH, displayed cytokine patterns indicative of a Th1 response significant quantities of IL-2 and IFN-gamma were produced while IL-4 and IL-10 secretion was insignificantly different from normal controls. The immunological phenotype of the AC-primed hosts could be altered by systemic treatment with antibodies against either a Th1 cytokine (IFN-gamma) or a Th2 cytokine (IL-10). Hosts treated with anti-IL-10 antibody and subsequently primed in the AC with ACAID-inducing antigens developed normal DTH responses, while hosts treated with anti-IFN-gamma antibody and primed in the AC with antigens that normally produce positive DTH responses failed to develop positive DTH collectively the results support the proposition that immune privilege in the AC of the eye is due to the selective activation of a Th2 population that cross-regulates Th1 responses.     

Decreased T-cell proliferation and skewed immune responses in LLC-bearing mice.

Deficits in immune cell responses have been reported in cancer patients. We used the murine Lewis lung carcinoma (LLC) model to better understand these deficits. The goal of this study was to determine if the immune responses of LLC tumor-bearing (TB) mice differ from control mice and whether the difference could be attributed to either antigen-presenting cells (FPC) or to T cells. Tumors were first allowed to grow in vivo for approximately 2 weeks. Splenocytes were then isolated for in vitro proliferation and cytokine release studies. The results showed a decrease in mitogen-stimulated proliferation by unfractionated splenocyte cultures from TB mice when compared to control mice in response to concanavalin A (Con A), a T-cell mitogen. Decreased responses were also observed when the APC spleen cell fraction from TB mice was cultured with normal T cells, although proliferation was more prominently reduced in cultures of TB T cells plus normal APC. Also, splenocytes from TB mice secreted significantly increased levels of IFN-gamma, IL-4, and IL-10. Admixing APC from control mice with TB T cells significantly decreased levels of IL-4 and IL-10 secretion as compared to the levels secreted by cocultures of TB T cells and TB APC. The decreased cytokine profile in the presence of normal APC despite the presence of TB T cells suggests that APC contributes to the immune dysfunction, including Th skewing of tumor bearers, possibly through their influence on T-cell expansion and cytokine production. Finally, our assessment of the APC population contributing to the observed immune dysfunction--i.e., dendritic cells or macrophages--showed that the proliferation of TB T cells was decreased regardless of the APC population with which they were cocultured. However, normal T-cell proliferation was only reduced by the addition of TB macrophages and not by the addition of TB dendritic cells. In conclusion, our results demonstrate that LLC TB mice have a skewed immune response characterized by a decreased proliferative response with both T cells and APC affected by the presence of tumor.     

Human Th1 and Th2 lymphocytes: their role in the pathophysiology of atopy

In human beings, as in mice, two distinct patterns of cytokine secretion have been defined among CD4+ helper T-cell clones. Human type 1 helper (Th1), but not type 2 helper (Th2), cells produce interleukin-2 (IL-2), gamma-interferon (IFN-gamma), and tumor necrosis factor-beta, whereas Th2, but not Th1, cells secrete IL-4 and IL-5, but not IL-2 or IFN-gamma. Other cytokines, such as IL-3, IL-6, GM-CSF, or TNF-alpha, are produced by both Th1 and Th2 cells. Th0 cells, a third Th subset, show combined production of Th1- and Th2-type cytokines. The different cytokine patterns are associated with different functions. In general, Th2 cells provide an excellent helper function for B-cell antibody production, particularly of the IgE class. On the other hand, Th1 cells are responsible for delayed type hypersensitivity reactions and are cytolytic for autologous antigen-presenting cells, including B cells. Most allergen- or helminth-antigen-specific human CD4+ T-cell clones exhibit a Th2 phenotype, whereas most clones specific for bacterial antigens show a Th1 profile. Allergen-specific Th2 cells seem to play a crucial role in atopy. These cells induce IgE production via IL-4 and favor the proliferation, differentiation, and activation of eosinophils via IL-5. In addition, Th2-derived IL-3 and IL-4 are mast-cell growth factors that act in synergy, at least in vitro. Recent evidence indicates that allergen-specific Th2 cells are selectively enriched in tissues affected by allergic inflammation, such as the bronchial mucosa of subjects with allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of boar seminal immunosuppressive fraction on production of cytokines by Concanavalin A-stimulated spleen cells and on proliferation of B lymphoma cell lines

PROBLEM: The immunosuppressive fraction (ISF) of boar seminal vesicle fluid has recently been demonstrated to inhibit mitogen-stimulated proliferation of lymphocytes and antibody response to corpuscular and soluble antigens. The effects of ISF on in vitro and in vivo production of cytokines as well as its possible inhibitory effect on proliferation of B lymphoma cells remain to be elucidated. METHODS: The effect of ISF on proliferation of normal mouse spleen cells stimulated by Concanavalin A (Con A) and on mouse B lymphoma cells was measured by 3H-thymidine incorporation. Cytokines were determined in the supernatants of mouse spleen cells stimulated with Con A in the presence or absence of ISF by enzyme-linked immunosorbent assay (ELISA). In vivo cytokine production in the sera samples of mice treated with ISF and immunized with keyhole limpet hemocyanin (KLH) was followed by ELISA, too. RESULTS: We confirmed the inhibitory effect of ISF on Con A-stimulated lymphocyte proliferation. ISF affected cytokine production in the Con A-stimulated spleen cells: production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was lowered, but production of IL-4, IL-6, and IL-10 was enhanced. Similarly, in the sera samples of mice immunized with keyhole limpet hemocyanin (KLH), IL-2 and IFN-gamma levels were decreased by ISF. ISF inhibited proliferation of Ag 8 and X 63-IL-2 B lymphoma cells as well. CONCLUSIONS: ISF inhibited production of T helper1 (Th1) cytokines (IL-2 and IFN-gamma) and enhanced production of Th2 cytokines (IL-4, IL-6, and IL-10). ISF seems to shift the Th1/Th2 pattern in favor of Th2. ISF exhibited an antiproliferative activity on mouse B lymphoma cells.