Francisella tularensis-induced in vitro gamma interferon, tumor necrosis factor alpha, and interleukin 2 responses appear within 2 weeks of tularemia vaccination in human beings.

Cell-mediated immunity is essential for protection against the intracellular bacterium Francisella tularensis, which causes tularemia. Positive in vitro T-cell responses in the form of lymphocyte proliferation and lymphokine interleukin 2 (IL-2) and gamma interferon (IFN-gamma) secretion are found in memory immunity. Studies on the secretion of lymphokines with regard to the developing immunity to F. tularensis have not been published. Therefore, 14 subjects with no clinical history of tularemia were vaccinated with a live F. tularensis vaccine strain. The in vitro responses of five subjects (antigen-induced mononuclear cell and whole blood culture DNA synthesis and cytokine secretion) were measured twice a week throughout the period from 0 to 35 days after vaccination, and the peripheral blood lymphocyte subpopulations of nine subjects were determined between days 0 and 14. Positive reactions, i.e., responses exceeding those on day 0, were reached on day 10 with regard to the whole blood culture DNA synthesis response and IL-2 and IFN-gamma secretion and on day 14 with regard to the mononuclear cell DNA synthesis response and tumor necrosis factor alpha (TNF-alpha) secretion. No measurable IL-4 was found in either the immune or nonimmune supernatants. Since the secretion of TNF-alpha was related to immunization, this points to the specificity of the phenomenon, even though the type of secreting cell is not yet known. If it is shown later that specific T cells produce it, the TNF-alpha response and the negative IL-4 finding may speak for the importance of the Th1-like pattern in immunity to F. tularensis.     

IL-13 production by allergen-stimulated T cells is increased in allergic disease and associated with IL-5 but not IFN-gamma expression.

Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper type 2 (Th2) cytokine IL-4, but its role in allergen-driven T-cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non-atopic controls results in elevated IL-13 synthesis in the context of a 'Th2-type' pattern. Freshly isolated peripheral blood mononuclear cells (PBMC) obtained from sensitized atopic patients with allergic disease, and non-atopic control subjects, were cultured with the allergens Phleum pratense (Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non-allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL-13, along with IL-5 and interferon-gamma (IFN-gamma) (Th2- and Th1-type cytokines, respectively) were determined by enzyme-linked immunosorbent assay (ELISA). Allergen-induced IL-13 and IL-5 production by T cells from patients with allergic disease was markedly elevated (P = 0.0075 and P = 0.0004, respectively) compared with non-atopic controls, whereas IFN-gamma production was not significantly different. In contrast to allergen, the prototypic Th1-type antigen M. tuberculosis PPD induced an excess of IFN-gamma over IL-13 and IL-5 production, and absolute concentrations of cytokines were not affected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN-gamma or IL-12, cytokines known to inhibit Th2-type responses, significantly inhibited allergen-driven production of both IL-13 and IL-5, but not T-cell proliferation, whereas exogenous IL-4 did not significantly affect production of IL-13 or IL-5. We conclude that allergen-specific T cells from atopic subjects secrete elevated quantities of IL-13 compared with non-atopic controls, in the context of a Th2-type pattern of cytokine production.     

Nickel antigen induces IL-2 secretion and IL-2 receptor expression mainly on CD4+ T cells, but no measurable gamma interferon secretion in peripheral blood mononuclear cell cultures in delayed type hypersensitivity to nickel.

Nickel sulphate antigen-induced peripheral blood lymphocyte activation in vitro was characterized by lymphokine measurement (IL-2, IFN-gamma) and phenotyping of the IL-2 responsive cells. Mononuclear cells from nickel-sensitive patients synthesized more DNA, produced more IL-2 and had more IL-2 receptor positive cells in response to nickel than did those of the control subjects. On the other hand no IFN-gamma was detectable in the nickel supernatants, while PPD, used as the control antigen, induced pronounced quantities of IFN-gamma with an equal amount of DNA synthesis. The increase in IL-2 receptor positive cells was due to activation of CD4+ (helper/inducer) T cells. T cells with HLA-DR antigen surface markers were more numerous on each day of culture than cells with IL-2 receptors. These two activation markers were co-expressed on the same cells only to a certain extent, thus perhaps reflecting different types or phases of activation. In conclusion, nickel-induced peripheral blood mononuclear cell activation in vitro differs from microbial antigen-induced activation with respect to its modest or non-existent IFN-gamma response.     

IL-4 acts as a homeostatic regulator of IL-2-induced TNF and IFN-gamma.

Interleukin-4 (IL-4) is a cytokine secreted by interleukin-2 (IL-2)-activated lymphocytes. IL-2-stimulated lymphocytes also secrete two cytokines, tumour necrosis factor (TNF) and gamma-interferon (IFN-gamma), which contribute to effector function and which may themselves recruit fresh, cytokine-secreting effector cells. We have now investigated whether the IL-4 induced is able to homeostatically regulate secretion of the TNF and IFN-gamma. Peripheral blood mononuclear cells or lymphocytes from normal donors and from patients with neoplastic disease were cultured in the presence of IL-2 alone, IL-4 alone or with both cytokines. IL-2 induced high levels of TNF and IFN-gamma secretion in both groups. The addition of recombinant IL-4 to these IL-2-stimulated cultures lead to significant inhibition of IFN-gamma and TNF production. IFN-gamma secretion was reduced by 50-99% in normal donors and by between 11% and 99% in patients (P less than 0.001). TNF levels induced by IL-2 were similarly reduced by IL-4 both in normal donors (P less than 0.003) and in patients (P less than 0.01). These inhibitory effects were produced by IL-4 at doses of IL-2 attainable in vivo. Inhibition appears to represent a homeostatic regulatory mechanism which may limit recruitment of fresh activated killer (AK) cells. When endogenous IL-4 activity in IL-2-activated lymphocytes was blocked by anti-IL-4 antibody, significantly higher levels of IFN-gamma and TNF were secreted (P less than 0.05). Since both TNF and IFN-gamma may contribute to the anti-neoplastic action of IL-2, manipulating the level of IL-4 activity in vivo could augment the benefits of IL-2 immunotherapy.     

The skin fungus-induced Th1- and Th2-related cytokine, chemokine and prostaglandin E2 production in peripheral blood mononuclear cells from patients with atopic dermatitis and psoriasis vulgaris

BACKGROUND: It is suggested that skin fungi may be involved in the development of atopic dermatitis (AD) and psoriasis vulgaris (PV). OBJECTIVE: We studied skin fungus-induced Th1- or Th2-related cytokine, chemokine and prostaglandin E2 (PGE2) secretion in peripheral blood mononuclear cells (PBMC) from patients with AD and PV and normal subjects. METHODS: PBMC were cultured with the extracts of Malassezia furfur (MF), Candida albicans (CA) and Trichophyton rubrum (TR). The cytokine, chemokine and PGE2 amounts in the supernatants were measured by enzyme-linked immunosorbent assays. RESULTS: MF induced IL-4 and macrophage-derived chemokine (MDC) secretion in AD patients, while induced IFN-gamma and interferon-inducible protein of 10 kDa (IP-10) secretion in PV patients, however, did not induce either secretion in normal subjects. CA induced IL-4, MDC, IFN-gamma and IP-10 secretion in AD and PV patients and normal subjects. In AD patients, the magnitude of IL-4 and MDC responses to CA was higher than that to MF. Compared with PV patients and normal subjects, the magnitude of IL-4 and MDC responses to CA was higher while that of IFN-gamma and IP-10 responses to CA was lower in AD patients. TR induced moderate IL-4 and MDC secretion only in AD patients. The three fungi induced higher levels of PGE2 secretion in AD patients than in PV patients and normal subjects. Cyclooxygenase-2 inhibitor NS-398 suppressed PGE2 responses to MF, CA and TR, and partially suppressed IL-4 and MDC responses to MF, CA and TR, while enhanced IFN-gamma and IP-10 responses to CA in AD patients, and these effects of NS-398 were reversed by cyclic AMP analogue. CONCLUSION: AD patients manifest Th2-skewed responses to MF, CA and TR, which may be partially attributable to the enhanced PGE2 responses to these fungi. PV patients manifest Th1-skewed responses to MF.     

The antibody response to HBs antigen is regulated by coordinated Th1 and Th2 cytokine production in healthy neonates

A proportion of healthy neonates fail to produce protective levels of anti-HBs antibody following vaccination with recombinant hepatitis B vaccine. This study was undertaken to investigate contribution of Th1 and Th2 responses to anti-HBs antibody production and to explore the mechanism(s) of unresponsiveness to HBsAg in human neonates. Peripheral blood manonuclear cells (PBMCs) were isolated form 28 nonresponder (anti-HBs antibody <10 IU/l) and 25 responder neonates. The cells were stimulated in vitro with recombinant HBsAg and PHA mitogen and concentrations of IL-4, IL-10 and IFN-gamma were quantified in culture supernatants by sandwich ELISA. Our results demonstrated significantly increased production of all cytokines, including IL-4 (P < 0.001), IL-10 (P < 0.002) and IFN-gamma (P < 0.01) in responder compared to nonresponder vaccinees. No significant differences, however, were observed between the two groups of neonates in the levels of cytokines induced by PHA or secreted in absence of antigen and mitogen. Our findings suggest that unresponsiveness to recombinant HBsAg in healthy neonates is linked to inadequate secretion of both Th1 and Th2 cytokines.     

Diminished interleukin 2 production and receptor generation characterize the acquired immunodeficiency syndrome

The Acquired Immunodeficiency Syndrome (AIDS) is a disease found primarily in homosexual men, consisting of opportunistic infections and tumors, and is due to an acquired T-cell defect. In the present report, we studied various T-cell functions which might serve to distinguish homosexuals with a symptom complex including lymphadenopathy from those with AIDS. T lymphocytes from the lymphadenopathy and AIDS patients had markedly depressed proliferative responses in the autologous (auto) and allogeneic (allo) mixed lymphocyte reaction (MLR) compared to healthy homosexuals or heterosexual controls (P less than 0.001). Since proliferation in the MLR depends upon interleukin 2 (IL-2), a T-cell growth factor, we studied the production of and response to IL-2 in various groups of homosexuals and heterosexual controls. IL-2 production was markedly depressed in the lymphadenopathy and AIDS patients, 1.0 and 0.1 U/ml, respectively, compared to the healthy homosexual or heterosexual controls, both 5.0 U/ml (P less than 0.05 and P less than 0.01, respectively). Although the auto MLR of the lymphadenopathy patients rose to control values with the addition of exogenous IL-2, the auto MLR of the AIDS patients did not (P less than 0.01). This lack of responsiveness to IL-2 in the AIDS group was due to their inability to generate IL-2 receptors as shown by the absence of IL-2 absorption by activated cells and the absence of the Tac antigen (IL-2 receptor) on these same cells. The T4+ and T8+ T-cell subsets from the AIDS patients each demonstrated depressed IL-2 production and responsiveness following activation with autologous cells or mitogen, as well as the absence of Tac antigen. The diminished T-cell proliferation in the auto MLR in the lymphadenopathy group is associated with one defect, low IL-2 production, while the depressed proliferation in the AIDS group is associated with two defects, low IL-2 production and a lack of IL-2 receptor generation. These studies demonstrate that IL-2 receptor generation helps distinguish homosexuals with lymphadenopathy from those with AIDS, and that in addition to T-cell defects in the OKT4+ T-cell subset there are significant abnormalities in the OKT8+ T-cell subset in AIDS patients.     

Study of activated T cells in man. II. Interleukin 2 receptor and transferrin receptor expression on T cells and production of interleukin 2 in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex

Because the expression of interleukin 2 (IL-2) receptor and transferrin receptor is essential for the proliferation of T cells to mitogens and antigens, we examined the expression of monoclonal antibody defined IL-2 receptor (Tac antigen) and transferrin receptor on unstimulated as well as on phytohemagglutinin (PHA)-activated highly enriched T cells from patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC). A trend of increased proportion of unstimulated T cells with Tac antigen and transferrin receptor was observed in patients with AIDS and ARC when compared to healthy heterosexual controls, but the differences were not significantly (P greater than 0.1). The proportions of Tac+ PHA-activated T cells were, however, significantly decreased in AIDS (P less than 0.001). ARC (P less than 0.001), and asymptomatic homosexuals (P less than 0.01) when compared to healthy heterosexuals. The proportions of transferrin receptor positive PHA-activated T cells were not significantly different among various groups. A significantly (P less than 0.01) decreased production of IL-2 was observed in AIDS. This study suggests that the poor proliferative responses of T cells may be due to several defects in lymphocyte-cytokine cascade and the deficiency of Tac antigen expression and of the production of IL-2 could be a few of several abnormalities contributing to poor T-cell functions in AIDS.     

Effects of probiotic bacteria and their genomic DNA on TH1/TH2-cytokine production by peripheral blood mononuclear cells (PBMCs) of healthy and allergic subjects

Among the factors potentially involved in the increased prevalence of allergic diseases, modification of the intestinal flora or lack of microbial exposure during childhood has been proposed. T(H)2-cytokines increase the production of IgE and stimulate mast cells and eosinophils, whereas T(H)1-cytokines, such as IFN-gamma, may suppress IgE synthesis and stimulate the expression of the secretory piece of IgA. Thus, a dysregulation in the expression of T(H)1- and T(H)2-cytokines may contribute to the initiation and maintenance of allergic diseases. Lactobacilli belonging to the natural intestinal microflora were reported to reduce the incidence of atopic dermatitis and the severity of allergic manifestations and to modulate T(H)1/T(H)2 responses. The mechanisms still remain to be elucidated. We sought to assess the effect of different probiotics, Lactobacillus rhamnosus GG, Lactobacillus gasseri (PA16/8), Bifidobacterium bifidum (MP20/5), and Bifidobacterium longum (SP07/3), on the T(H)1 and T(H)2 responses of peripheral blood mononuclear cells (PBMCs) from healthy subjects and from patients with allergy against house dust mite to Staphylococcus enterotoxin A (SEA) and Dermatophagoides pteronyssinus (Dpt). To elucidate the molecular basis of these effects, the effects of bacterial genomic DNA were compared with the effects of viable bacteria. PBMCs from allergic patients and from healthy donors were incubated for 24 or 48 h, respectively, with or without SEA and Dpt allergens. The effects of preincubation with live probiotic bacteria and the effect of their genomic DNA, added simultaneously to cultures and incubated for 24h, were assessed by measuring T(H)1/T(H)2-cytokine production. The tested live Gram-positive probiotic bacteria and their genomic DNA inhibited SEA- and Dpt-stimulated secretion of T(H)2-cytokines (IL-4 and IL-5) and enhanced the stimulation of IFN-gamma. This effect was dose-dependent with a dosage-optimum, which was identical for all lactic acid producing bacteria (LAB) tested (10 bacteria per PBMC) and their DNA (75 ng/ml). Based on the maximal effects achieved with LAB and their DNA, more than 50% of the effects seem to be contributed by DNA. No significant effect was induced by the control, Gram-negative Escherichia coli TG1. Lactobacilli and bifidobacteria reduced SEA-stimulated IL-4 and IL-5 production more effectively in PBMCs from healthy subjects than from allergic patients. In contrast to this, inhibition of Dpt-stimulated IL-4- and IL-5-secretion was more pronounced in cells from allergic subjects. Compared with living LAB, bacterial DNA inhibited IL-4- and IL-5-secretion in a similar manner. SEA- and even more so Dpt-stimulated IFN-gamma stimulation by living LAB was less pronounced in allergic than in healthy subjects, whereas IFN-gamma stimulation by their DNA was more pronounced in allergic subjects. The tested probiotic bacteria as well as their genomic DNA modulated the T(H)1/T(H)2 response to some allergens dose-dependently. DNA seems to contribute to 50% of the effect exerted by living bacteria in this in vitro model. The magnitude of the probiotic effects differed between healthy and allergic subjects. Whether the modulation found for the tested strains might be useful for the prevention and treatment of allergic diseases has to be assessed in clinical trials.     

In vitro immunomodulatory effects of interleukin-2 and thymosin fraction V in acquired immune deficiency syndrome

A monoclonal antibody (anti-Tac) that appears to bind the receptor for interleukin-2 (IL-2) was used to quantitate lymphocytes that express IL-2 receptors (IL-2R) in response to phytohemagglutinin (PHA) stimulation. 62 +/- 4% of the cells expressed IL-2R in response to PHA in twelve normal subjects compared to 22 +/- 4% in fourteen patients with acquired immune deficiency syndrome (AIDS) (P less than 0.001) and 50 +/- 6% in six patients with AIDS-related complex (P less than 0.1). There was no effect on IL-2R expression, when lymphocytes from seven controls were incubated with IL-2 (20 mu/ml) or thymosin fraction V (10 mu/ml) for 72 h. However, when the lymphocytes from seven patients with AIDS were incubated with IL-2, the IL-2R rose from 18 +/- 3% to 31 +/- 3% (P less than 0.005) and with a fraction V to 29 +/- 3% (P less than 0.001). In addition, IL-2 augmented the PHA-induced proliferative responses in patients with AIDS-related complex and AIDS and normal controls, whereas thymosin fraction V had no significant effect. Thymosin fraction V also enhanced the IL-2 production of PHA-stimulated mononuclear cells obtained from six patients with AIDS and six normal controls. These results suggest that both IL-2 and thymosin fraction V can modulate in vitro T-cell function in patients with AIDS.     

T-cell abnormalities in inflammatory bowel disease are mediated by interleukin 2

Inflammatory bowel disease (IBD) may be an immunologically mediated disorder in which T cells are unable to respond appropriately to cell surface-associated antigens. To test this possibility, 37 patients with IBD, 24 with Crohn's disease and 13 with ulcerative colitis who were not being treated with immunosuppressive therapy were studied. The ability of T cells to proliferate in response to autologous or allogeneic cells, i.e., the autologous or allogeneic mixed-lymphocyte reaction (MLR) was tested. The autologous MLR was depressed using patient cells compared to control cells, regardless of disease type or activity (1564 +/- 223 cpm versus 3300 +/- 381 cpm, P less than 0.05) while the allogeneic MLR was depressed in patients with active disease only (29,833 +/- 2871 cpm versus 46,799 +/- 3340 cpm, P less than 0.01). The ability of T cells to recognize and lyse allogeneic cells, allogeneic cell-mediated lympholysis (CML), was also low in patients with active disease (24 +/- 4% versus 37 +/- 3%, P less than 0.05). Since T-cell proliferation and cytotoxicity depend upon adequate production of and response to a T-cell growth factor, interleukin 2 (IL-2), IL-2 production and responsiveness in IBD were studied. IL-2 production by patient T cells in response to phytohemagglutinin was only 39% of control values, P less than 0.05. The response to IL-2 was measured by the increase in T-cell proliferation in the autologous MLR in medium alone or medium supplemented with IL-2. Control T-cell proliferation rose from 3300 +/- 381 cpm to 10,761 +/- 428 cpm with exogenous IL-2 (P less than 0.001). Patient T-cell proliferation rose from 1564 +/- 223 cpm to 6817 +/- 771 cpm with IL-2 (P less than 0.001) but did not reach the level of the IL-2-supplemented control autologous MLR (P less than 0.05). In addition, the percentage of activated patient T cells having Tac antigen (IL-2 receptor) was depressed (P less than 0.05). These findings did not vary with disease type or activity. It is concluded from these data that peripheral blood T lymphocytes from patients with IBD have a diminished response to cell surface antigens which is associated with a decrease in IL-2 production and receptor generation. These defects may be responsible for the depressed T-cell proliferation and cytotoxicity that accompany IBD.     

Functional heterogeneity among CD4+ T-cell clones from blood and skin lesions of leprosy patients. Identification of T-cell clones distinct from Th0, Th1 and Th2.

In the present study we examined the functional properties of T-cell clones reactive with Mycobacterium leprae and other mycobacterial antigens. Clones isolated from the skin lesions and blood of leprosy patients across the spectrum were exclusively CD4+CD8- and expressed the alpha beta T-cell receptor. Substantial heterogeneity in the production of cytokines, in particular interleukin-4 (IL-4), was observed, although no striking correlation with clinical status was apparent. A variety of patterns of cytokine secretion distinct from those of T-helper type-1 (Th1) Th2 or Th0, as defined in murine studies, was evident. Most noteworthy was a large number of clones from skin which secreted neither IL-2 nor IL-4, but large amounts of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Clones isolated from the blood of leprosy patients had a more restricted cytokine secretion profile, and appeared to resemble more closely previously described patterns, including those of high level production of IL-2 and/or IL-4. Virtually all clones, from either skin or blood, produced high levels of IFN-gamma, and thus many clones were IL-4 and IFN-gamma co-producers. The pattern of cytokine production by skin-derived T-cell clones was significantly affected by the in vitro activation status of the cells. Cells enriched in activated blasts tended to produce more IL-4 than small resting cells. In addition, the production of IFN-gamma by skin T-cell clones after < or = 10 weeks of culture was strikingly distinct from that of these clones after 5 months of culture. IL-4 and IFN-gamma co-producing clones shifted to a Th2-like pattern with much less IFN-gamma secretion, whereas non-IL-4-producing clones secreted much higher levels of IFN-gamma after prolonged culture, and became much more Th1-like. However, there was still no correlation between clinical status and pattern of cytokines produced. These results imply that a high fraction of T cells exists in leprosy lesions that is distinct from or that has not yet fully matured into Th1 or Th2 cells.     

Interleukin-2 in rheumatoid arthritis: production of and response to interleukin-2 in rheumatoid synovial fluid, synovial tissue and peripheral blood.

Several aspects of interleukin-2 (IL-2) generation and function were studied employing mononuclear cells from synovial fluid (SF), synovial tissue (ST) and peripheral blood (PB) of patients with rheumatoid arthritis (RA). Decreased PHA stimulated IL-2 production by lymphocytes from rheumatoid ST, SF (P less than 0.02), and PB (P less than 0.01) was observed when compared to normal blood and SF of patients with gout. The proliferative response of rheumatoid lymphocyte blasts exposed to exogenous IL-2 was also defective (P less than 0.05-0.001). This defect was greater in SF than in rheumatoid PB (P less than 0.05-0.001). In addition to the proliferative response, the effect of IL-2 on interferon-gamma (IFN-gamma) production was also examined. Rheumatoid lymphocytes from both PB and SF produced less IFN-gamma after overnight treatment with IL-2 than did normal PB lymphocytes. This decreased IFN-gamma induction was discordant with the excellent enhancement by IL-2 of natural killer activity. Removal of adherent cells in synovial fluid did not correct this deficit. Abnormalities in the biology of IL-2 and IFN-gamma suggest that impaired T cell function could contribute to the immunopathogenesis of RA.     

Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment.

A longitudinal investigation has been conducted into the cell-mediated immune responses of onchocerciasis patients after a single-dose treatment with ivermectin. Untreated patients tested for delayed cutaneous hypersensitivity (DCH) to seven recall antigens showed lower responses than infection-free control individuals (P less than 0.01), but 6 and 14 months after treatment DCH reactions increased to similar levels to those seen in the controls. The in vitro cellular reactivity to Onchocerca volvulus-derived antigen (OvAg) was reduced in untreated patients as compared with controls, and the lymphocyte blastogenic responses to OvAg and streptolysin-O clearly improved up to 14 months after treatment. Peripheral blood mononuclear cells (PBMC) from untreated patients produced IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and IL-6 in response to mitogenic stimulation with phytohaemagglutinin (PHA), only low levels of IL-1 beta, IL-2 and TNF-alpha in response to OvAg, but higher amounts of IL-4 and interferon-gamma (IFN-gamma) in response to OvAg than control individuals. After ivermectin treatment, the OvAg-induced production of IL-1 beta and TNF-alpha increased significantly 1 and 14 months after treatment. The PHA-induced production of IL-2 and IL-4 increased 1 month after treatment and remained significantly elevated until 14 months after treatment, whereas the OvAg-specific secretion of IL-2, IL-4 and IFN-gamma did not change after ivermectin treatment. Flow cytometric analysis of lymphocyte-subsets in the peripheral blood of untreated patients revealed a relative and absolute (P less than 0.01) diminution of CD4+ cells and a significantly smaller CD4+/CD8+ cell ratio as compared with controls. By 4 weeks after treatment and thereafter, CD4+ T cells increased relatively and absolutely (P less than 0.01); likewise there was an absolute increase in T-helper-inducer cells (CD4+CD45RO+) and a temporarily improved CD4+/CD8+ cell ratio (P = 0.001). The expression of the low-affinity receptor for IgE (CD23) on total lymphocytes decreased from 14% to 7% by 14 months after treatment. The CD8+ cells and CD3+TCR gamma delta + cells were higher in patients than in controls and both remained elevated until 14 months after treatment. These results suggest a distinctly improved cellular immunity in human onchocerciasis that was facilitated by ivermectin therapy.     

Proliferation and release of IL-5 and IFN-gamma by peripheral blood mononuclear cells from cat-allergic asthmatics and rhinitics, non-cat-allergic asthmatics, and normal controls to peptides derived from Fel d 1 chain 1.

BACKGROUND: In general, T cells from normal, nonatopic individuals respond to aeroallergens with synthesis and release of IFN-gamma. In contrast, release of T(H)2-type cytokines by activated lymphocytes is a feature of allergic rhinitis and atopic asthma. OBJECTIVE: The purpose of this study was to determine differences in T-cell recognition of epitopes within allergenic sequences, in terms of proliferation and cytokine production, in subjects with atopic asthma compared with subjects with allergic rhinitis and normal controls. METHODS: Proliferative responses and IL-5/IFN-gamma release patterns from PBMCs from cat-allergic asthmatic, cat-allergic rhinitic, and non-cat-allergic asthmatic subjects and nonatopic normal controls were determined in primary cultures. Cells were challenged with 7 overlapping peptides spanning chain 1 of the major cat allergen, Fel d 1. RESULTS: The 4 groups did not differ with respect to the ability to mount proliferative responses to Fel d 1 peptides. In all groups, the IFN-gamma responses were predominantly to the amino terminus peptides. Cat-allergic and non-cat-allergic asthmatic subjects (and not cat-allergic rhinitic subjects and normal controls) made IL-5 responses to most of the Fel d 1 peptides, the result being a mixed (T(H)0) cytokine response at the N-terminus and a restricted (T(H)2) response at the C-terminus. CONCLUSION: Proliferative and IL-5/IFN-gamma responses of T cells from asthmatic and atopic rhinitic subjects and normal controls to allergen peptides can be dissociated. Furthermore, differing cytokine responses to peptides derived from a single antigen suggest that certain domains of the molecule might preferentially induce IL-5 rather than IFN-gamma and as a result could be more important in disease pathogenesis.     

Blood lymphocyte proliferation, cytokine secretion and appearance of T cells with activation surface markers in cultures with Helicobacter pylori. Comparison of the responses of subjects with and without antibodies to H. pylori.

A whole inactivated H. pylori bacterium preparation was found to stimulate blood mononuclear cells from both antibody-positive and antibody-negative subjects, but the antibody-positive subjects tended to have lower proliferation responses. The present study was designed to characterize T cell activation further by measuring several components of the response. Eighty-seven subjects (80 dyspeptic patients and seven healthy persons from the laboratory staff) with or without antibodies to H. pylori were studied by measuring the DNA synthesis induced by several H. pylori concentrations (1-23 micrograms/ml) and the control stimulants PPD, tetanus toxoid and pokeweed mitogen (PWM). H. pylori-induced secretion of interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4), soluble CD8 and IL-2 receptor (IL-2R) molecules and H. pylori- and PPD-induced appearances of IL-2R+ and HLA-DR+ T cells were measured in a smaller number of subjects. H. pylori-induced DNA synthesis was again lower in the antibody/bacterium-positive subjects, while no differences between the two groups were found in cultures stimulated by unrelated antigens or PWM. Soluble IL-2R and TNF-alpha were detectable in cultures with H. pylori from all subjects, while the amount of IL-2 did not differ from that in the background culture. No differences were found in the amounts of IL-2 or soluble IL-2R between the antibody-positive and negative subjects; while the former tended to secrete more soluble CD8 molecules, a difference which was significant with the smaller H. pylori concentration used (P less than 0.01). The numbers of HLA-DR+ and IL-2R+ T cells increased in cultures with H. pylori or PPD from all the subjects, the majority of both cells having the CD4 phenotype. Numbers of DR+ and IL-2R+ T cells were similar in the cultures of the antibody-positive and negative subjects, but the respective CD8 subsets were increased in the former. The confirmed decrease in proliferation in the antibody-positive subjects does not seem to be connected with lower IL-2/IL-2R responses but may involve CD8 cell activation.     

Selective correlation of interferon-gamma, tumour necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor with immunoglobulin G1 and immunoglobulin G3 subclass antibody in leprosy.

Dysregulation of both B- and T-cell responses is observed in leprosy. Immunoglobulin G1 (IgG1) and IgG3 antibody subclasses are selectively elevated towards the lepromatous or disseminated form of the disease accompanied by a depression of T-cell responses. T-cell and macrophage cytokines influence antibody class switching, differentiation and proliferation of B cells. To understand the dynamic nature of the immune response in leprosy, we examined the relationship between circulating Mycobacterium leprae-specific antibodies and secreted cytokines [interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-5, IL-10, IL-6, tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF)] in leprosy patients (19 lepromatous patients; 25 tuberculoid patients) and their exposed household contacts (HC=14) in response to M. leprae antigens. Paired comparison revealed a highly significant negative correlation between IFN-gamma and IgG (P=0.016), IgG1 (P<0.001) and IgG3 (P=0. 007) antibodies. No significant relationship was observed with other T-cell cytokines (IL-2, IL-5 and IL-10). These results strongly suggest that IFN-gamma may play a role in down-regulating antigen-specific IgG1 and IgG3 antibodies. Among the macrophage cytokines, TNF-alpha and GM-CSF which have not been shown to play a role in B-cell activation were positively associated with IgG1 (TNF-alpha, P=0.0005; GM-CSF, P=0.001) and IgG3 (TNF-alpha, P=0.001; GM-CSF, P=0.021) antibodies. Since macrophages have high-affinity Fc receptors for IgG1 and IgG3, it is possible that antigen uptake via these receptors may influence cytokine expression of TNF-alpha, a key modulator of disease pathogenesis in mycobacterial diseases. We are currently investigating the role of Fc receptors on activated macrophages, in expression of pro-inflammatory cytokines in mycobacterial diseases.     

Frequency of measles virus-specific CD4+ and CD8+ T cells in subjects seronegative or highly seropositive for measles vaccine

The protective effect of measles immunization is due to humoral and cell-mediated immune responses. Little is known about cell-mediated immunity (CMI) to measles vaccine virus, the relative contribution of CD4(+) and CD8(+) T cells to variability in such immune responses, and the immunologic longevity of the CMI after measles vaccination in humans. Our study characterizes cellular immune response in subjects seronegative or highly seropositive for measles vaccine immunoglobulin G-specific antibody, aged 15 to 25 years, previously immunized with two doses of measles-mumps-rubella II vaccine. We evaluated the ability of subjects to respond to measles vaccine virus by measuring measles virus-specific T-cell proliferation. We examined the frequencies of measles virus-specific memory Th1 and Th2 cells by an ELISPOT assay. Our results demonstrated that proliferation of T cells in seronegative subjects was significantly lower than that for highly seropositive subjects (P = 0.003). Gamma interferon (IFN-gamma) secretion predominated over interleukin 4 (IL-4) secretion in response to measles virus in both groups. The median frequency of measles virus-reactive CD8(+) T cells secreting IFN-gamma was 0.09% in seronegative subjects and 0.43% in highly seropositive subjects (P = 0.04). The median frequency of CD4(+) T cells secreting IL-4 in response to measles virus was 0.03% in seronegative subjects and 0.09% in highly seropositive subjects (P = 0.005). These data confirm the presence of measles virus-specific cellular immune responses post-measles vaccine immunization in humans. The detection of measles virus-induced IFN-gamma and IL-4 production by ELISPOT can be used to identify measles virus-specific low-frequency memory T cells in subjects immunized with measles vaccine. These differences agree in directionality with the observed antibody response phenotype.     

Influence of CD28 co-stimulation on cytokine production is mainly regulated via interleukin-2.

Interaction of CD28 with its ligand B7 plays an important role in the initiation of immune responses. The co-stimulatory signal generated by cross-linking of CD28 molecules results in enhanced T-cell proliferation and augmentation of cytokine production. In particular, mRNA levels of T-helper 1 (Th1)-type cytokines, such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) are reported to be strongly increased. We investigated the effect of CD28 co-stimulation on the production of Th2-type cytokines. CD28 mAb induced a strong augmentation of IL-2 secretion in activated T-cell clones. Production of IFN-gamma was also enhanced, but the increase in IL-4 secretion was generally moderate. Augmentation of IL-4 production by CD28 was most pronounced in clones that produced low amounts of IL-2, compared to clones producing high levels of IL-2. It was found that the up-regulation of IL-4 by CD28 co-stimulation was mainly controlled indirectly via an increase of IL-2. Some clones could produce IL-4 in an IL-2-independent manner; in these situations CD28 co-stimulation had no augmenting effect on the production of IL-4. The secretion of IL-4 by peripheral blood CD4+ T cells, that were activated with B7-expressing transfectants, was also found to be dependent on IL-2. Finally, Northern blot analysis confirmed that co-stimulation of CD28 primarily affected IL-2 production, and that inhibition of IL-2/IL-2 receptor interaction abolished the augmenting action of CD28 monoclonal antibody on the production of the Th2-type cytokines IL-4, IL-5 and IL-10 and of the Th1 cytokine IFN-gamma.