The importance of CD54 and CD86 costimulation in T cells stimulated with Candida albicans and Dermatophagoides farinae antigens in patients with atopic dermatitis

A number of studies have demonstrated an increased frequency of allergen-specific T cells producing increased amounts of interleukin-4 (IL-4) and IL-5, but little interferon-y in both the peripheral blood and skin lesions of patients with atopic dermatitis (AD). In this study, to further clarify the characteristics of T cells obtained from AD patients, we examined the dependency of the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) from AD patients on costimulatory molecules. The antigens used were Candida albicans and Dermatophagoides farinae, for which AD patients show increased levels of IgE antibodies. PBMC from control healthy donors stimulated with these antigens incorporated [3H]-thymidine much more than PBMC from AD patients. The addition of anti-CD54, -CD40, -CD80 and -CD86 monoclonal antibodies to the cultures showed that the PBMC required only CD54 and CD86 for stimulation with C. albicans, but required CD54, CD80 and CD86 for stimulation with D. farinae. Among these monoclonal antibodies, the anti-CD54 antibody suppressed the proliferative responses of most PBMC, most effectively followed by the anti-CD86 antibody. However, there were no significant differences in the requirement for costimulatory molecules of PBMC proliferation stimulated with C. albicans or D. farinae between AD patients and healthy donors. Since many studies have suggested that T-helper type 1 and T-helper type 2 immune responses are different in their dependency on CD80 or CD86 costimulation, our present results suggest that the allergen-specific T cells of AD patients are not completely shifted to a T-helper type 2 subset.     

Evidence for a unique elastic sheath surrounding the vesicular arteries of the rabbit urinary bladder--studies of the microvasculature with microscopy and vascular corrosion casting

AD is associated with a bias of the T helper cells to show increased IL-4 and reduced interferon-gamma (IFN-gamma) production. The production of IFN-gamma and IL-4 and the development of Th cells into either high IFN-gamma or high IL-4 producers is strongly influenced by factors produced by antigen-presenting cells (APC), like IL-12 and prostaglandin E2 (PGE2). IL-12 selectively enhances IFN-gamma production and favours the development of IFN-gamma-producing Th cells, whereas PGE2 selectively inhibits IFN-gamma production by Th cells. The aim of this study was to test whether the increased IL-4/IFN-gamma production ratio by Th cells in AD can be explained by an increased PGE2/IL-12 production ratio by the APC. Monocytes were used as APC source. PGE2 and IL-12 production by lipopolysaccharide (LPS)-stimulated monocytes from 12 AD patients and 12 non-atopic controls was determined using two complementary experimental systems, whole blood cultures and purified monocytes. In addition, we determined IL-6 production as a measure of monocyte activation, and IL-10 production because IL-12 production by monocytes is highly influenced by endogenously produced IL-10. The monocytes from AD patients showed normal production levels of IL-6 and IL-10, a two-fold, but non-significant decrease in IL-12 production, and a significantly (three-fold) higher PGE2 production than those from non-atopic controls. Here we show for the first time that enhanced PGE2 production by monocytes in AD is not accompanied by a general rise in cytokine production. We conclude that AD is indeed associated with an increased PGE2/IL-12 production ratio by monocytes.     

Elevated serum levels of soluble CD30 in patients with atopic dermatitis (AD)

The immunopathology of AD is still unclear, but evidence for an immune response polarized towards Th2 activity has been provided. The CD30 molecule belongs to the tumour necrosis factor (TNF) receptor family and is expressed on activated T cells with a sustained expression in Th2 cells. This molecule also exists in a soluble form (sCD30). Elevated serum levels of sCD30 have been found in patients with Hodgkin's disease, chronic hepatitis B infection and HIV infection. Studies were undertaken to compare the serum levels of sCD30 in patients with AD (n=49) and healthy non-atopic controls (n=94). The presence of sCD30 was analysed with ELISA. A significantly higher concentration of sCD30 was noted in AD patients, median sCD30 level 29 U/ml (range 1-708 U/ml), compared with healthy non-atopic controls (P<0.001), where the median level was 11 U/ml with a range of 1-1042 U/ml. No correlation was found between sCD30 levels and total serum IgE, or between the AD patients' SCORAD values and concentration of sCD30. sCD30 levels were also analysed in 20 AD patients, which during ketoconazole treatment had improved their clinical scores and reduced their serum IgE and eosinophil cationic protein levels. However, no significant decrease in sCD30 levels was noted after treatment. The results show that patients with AD have elevated levels of sCD30, but without correlation to total serum IgE or disease activity.     

Allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in chronic skin lesions of atopic dermatitis

The majority of allergen-specific T cells derived from inhalant allergen patch test lesions in patients with atopic dermatitis were previously found to produce a restricted type-2 cytokine pattern. Recent studies, however, have revealed that in chronic eczematous skin lesions of patients with atopic dermatitis, expression of the type-1 cytokine interferon-gamma predominates. To evaluate cytokine production by allergen-specific T cells in chronic atopic dermatitis, we established house dust mite (Dermatophagoides pteronyssinus)-specific T-cell clones from the dermis of chronic skin lesions of sensitized adult patients with atopic dermatitis. Frequencies of skin-derived T cells proliferating in the presence of Dermatophagoides pteronyssinus were between one in 138 and one in 4255, indicating that only a minority of skin-infiltrating T cells are allergen specific. When these cells were analyzed for their capacity to produce interferon-gamma, the majority (71%) of these cells were found to express interferon-gamma mRNA and to secrete interferon-gamma protein, either alone or in combination with interleukin-4. Phenotypic analysis revealed that 15% of skin-infiltrating allergen-specific T cells were CD8+. No selection of Vbeta elements was detected in Dermatophagoides pteronyssinus-specific T-cell clones. These studies demonstrate that allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in lesional atopic dermatitis. The notion that the majority of allergen-specific, skin-infiltrating T cells are capable of producing interferon-gamma further supports the concept that interferon-gamma expression has major pathogenetic relevance for the chronic phase of atopic dermatitis.     

IgE hyperproduction through enhanced tyrosine phosphorylation of Janus kinase 3 in NC/Nga mice, a model for human atopic dermatitis

IgE hyperproduction frequently observed in patients with atopic dermatitis (AD) may greatly contribute to the pathogenesis of AD, but its mechanisms are still unclear. NC/Nga mice raised in nonsterile circumstances spontaneously suffered from AD-like skin lesions with elevation of plasma IgE levels. We investigated mechanisms of the IgE hyperproduction in NC/Nga mice. Splenic T cells from SPF NC/Nga mice had a level of CD40 ligand (CD40L) expression comparable to that of BALB/c mice. Although there was no difference in the expression of CD40 on B cells between NC/Nga and BALB/c mice, B cells of NC/Nga mice produced much more IgE in the presence of soluble CD40L and IL-4. The stimulation with CD40L and/or IL-4 resulted in tyrosine phosphorylation of Janus kinase 3 (JAK3) in B cells, which was more strongly inducible in NC/Nga mice than in BALB/c mice. In B cells isolated from PBMC of AD patients with high serum IgE levels, JAK3 was constitutively phosphorylated at the tyrosine residue, and its phosphorylation was enhanced by the treatment with CD40L and/or IL-4 as was that in splenic B cells of NC/Nga mice with dermatitis and high IgE levels. Thus, it is suggested that constitutive and enhanced JAK3 phosphorylation in B cells highly sensitive to CD40L and IL-4 may be attributable to IgE hyperproduction in NC/Nga mice and patients with AD.     

Evidence for superantigen involvement in skin homing of T cells in atopic dermatitis

The environmental factors that contribute to the homing of T cells in skin disease is unknown. The skin lesions of atopic dermatitis (AD) are frequently colonized with superantigen (SAg), producing strains of Staphylococcus aureus. In vitro, these superantigens have the capacity to activate and expand T cells expressing specific T cell receptor BV gene segments, and also to increase their skin homing capacity via upregulation of the skin homing receptor, cutaneous lymphocyte-associated antigen (CLA). These activities have been proposed to enhance the chronic cutaneous inflammation of AD, but an in vivo role for SAg has not been conclusively demonstrated. In this study, we sought direct evidence for in vivo SAg activity by comparing the SAg profiles of S. aureus cultured from the skin of AD subjects to the T cell receptor Vbeta repertoire of their skin homing (CLA+) T cells in peripheral blood. SAg secreting S. aureus strains were identified in six of 12 AD patients, and all of these subjects manifested significant SAg-appropriate Vbeta skewing within the CLA+ subsets of both their CD4+ and their CD8+ T cells. T cell receptor Vbeta skewing was not detectable among the overall CD4+ or CD8+ T cell subsets of these subjects, and was not present within the CLA+ T cell subsets of five patients with plaque psoriasis and 10 normal controls. T cell receptor BV genes from the presumptively SAg-expanded populations of skin homing T cells were cloned and sequenced from three subjects and, consistent with a SAg-driven effect, were found to be polyclonal. We conclude that SAg can contribute to AD pathogenesis by increasing the frequency of memory T cells able to migrate to and be activated within AD lesions.     

Effects of reconstructive surgery for left ventricular anterior aneurysm on ventriculoarterial coupling

Microbial agents are known to play a significant role in aggravating allergic diseases. Recently described viral and bacterial superantigens represent one important strategy by which infectious agents can stimulate the immune response. In previous work, we reported that the staphylococcal toxin toxic shock toxin-1 (TSST-1), a prototypic superantigen, induces in vitro total IgE synthesis after cross-linking T and B cells. This study was carried out to establish a potential link between superantigens and the enhanced IgE response to specific allergens in allergic patients. Peripheral blood mononuclear cells from atopic patients were isolated during and outside the pollen allergen season and stimulated with TSST-1, a prototypic superantigen. Total IgE and interferon-gamma production were measured in supernatants of these cultures. Outside the pollen season, TSST-1 significantly increased total IgE production only in the presence of exogenous interleukin-4, whereas during the pollen season IgE production was significantly enhanced without the need of exogenous interleukin-4. This increase in the absence of exogenous interleukin-4 was associated with significantly lower interferon-gamma production by peripheral blood mononuclear cells stimulated by TSST-1 during the pollen season. Moreover, TSST-1 stimulation of peripheral blood mononuclear cells from inhalant allergic patients was followed by an increased production of allergen-specific IgE that was restricted to the allergen to which the patient was allergic and recently exposed. In addition, TSST-1 induced on B cells the expression of B7.2, a molecule that has recently been demonstrated to enhance T helper 2 responses and to be involved in IgE regulation. This study, by demonstrating that superantigens can augment allergen-specific IgE synthesis and B7.2 expression, provides a mechanism by which microbial superantigens may modulate allergic responses.     

In vivo relevance of CD30 in atopic dermatitis

CD30 expression was evaluated by immunohistochemistry in lesional skin biopsies of eight patients with active atopic dermatitis (AD) and three patients with allergic contact (nickel-induced) dermatitis (ACD). CD30 expression was also assessed in a large panel of CD4+ and CD8+ T-cell clones generated from the skin biopsies of four patients with AD. Finally, the levels of soluble CD30 (sCD30) were measured in the serum of 41 patients with AD, 19 patients with ACD, and 60 healthy controls. In all specimens of lesional AD skin, where the great majority of infiltrating cells were CD4+ T cells, remarkable numbers of cells were CD30+, whereas virtually no CD30+ cells were found in the skin of patients with ACD. In CD4+ T-cell clones generated from the lesional AD skin, most of which produced both interleukin (IL)-4 and interferon-gamma (IFN-gamma) (Th0-like cells) or IL-4 and IL-5, but not IFN-gamma (Th2-like cells), CD30 expression directly correlated with the ability to produce IL-4 and IL-5, but was inversely related to IFN-gamma production. High levels of sCD30 (correlated with disease activity: r = 0.618) were detected in the serum of most AD patients, whereas there was no increase of sCD30 levels in the serum of patients with ACD. These data support the view that Th0/Th2-type responses predominate in the skin of patients with AD and suggest that the presence of CD30+ T cells in tissues and/or increased levels of sCD30 in biologic fluids are indicative of Th2-dominated responses.     

Cloning of the genes encoding two murine and human cochlear unconventional type I myosins

The predominance of Th2 cytokine-secreting pattern in allergic asthma has been known as a cause and an accelerating factor, and Th1 suppresses these allergic phenomena, but the role of Th0 clones is obscure. Because Th1/Th2 differentiation has been determined by cytokine environment, we investigated how mite-specific helper T cells stimulated in different cytokine environments actually influenced IgE and IgG4 synthesis, which are known to be regulatory immunoglobulins for allergic response. Th0 clones, which were mainly established in the presence of IL-12, provided a great deal of help for IgG4 and IgG1 synthesis, but did not provide help for IgE synthesis, whereas Th2 clones helped IgE synthesis prominently, and IgG4 and IgG1 synthesis marginally. These characteristics of Th0 clones were also true for Th0 clones obtained from patients who were successfully treated with desensitization therapy. Furthermore, the differences in helper activity between Th0 and Th2 clones were not ascribed solely to soluble factors. These data indicate that IgE and IgG4 synthesis is differentially regulated by antigen-specific T cells, and that conversion or selection from Th2 to Th0 by the addition of IL-12 may exhibit therapeutic effects.     

Local expansion of allergen-specific CD30+Th2-type gamma delta T cells in bronchial asthma

BACKGROUND: T lymphocytes infiltrating airways during the allergic immune response play a fundamental role in recruiting other specialized cells, such as eosinophils, by secreting interleukin 5 (IL-5), and promoting local and systemic IgE synthesis by producing IL-4. Whether these presumed allergen-specific T cells are of mucosal or systemic origin is still a matter of conjecture. MATERIALS AND METHODS: Immunophenotype, IL-4 production, and in vitro proliferative response to specific or unrelated allergens were analyzed in the bronchoalveolar lavage (BAL) fluid lymphocyte suspensions obtained from untreated patients with allergic asthma. Healthy subjects and patients affected by pulmonary sarcoidosis, a granulomatous lung disease characterized by infiltrating Th1 CD4+ lymphocytes, served as controls. RESULTS: The proportions of gamma delta T lymphocytes, mostly CD4+ or CD4- (-)CD8-, was higher in asthmatic subjects than in controls (p < 0.05). Most BAL gamma delta CD4+ lymphocytes of asthmatic patients displayed the T cell receptor (TCR)-gamma delta V delta 1 chain. While CD30 antigen coexpression on the surface of BAL alpha beta(+) T lymphocytes was low (ranging from 5 to 12%), about half of pulmonary gamma delta T cells coexpressed it. These cells produced IL-4 and negligible amounts of interferon-gamma (IFN gamma), and proliferated in vitro in response to purified specific but not unrelated allergens. In contrast, control or sarcoidosis gamma delta T cells never displayed the CD30 surface molecule or produced significant quantities of IL-4. CONCLUSIONS: These findings not only confirm our previous hypothesis that the allergen-specific Th2-type lymphocytes found in the lungs of asthmatic patients are gamma delta T cells belonging to airway mucosal immunocytes, but also strongly support the notion that asthma is a local rather than a systemic disease.     

A role for T-helper type 1 and type 2 cytokines in the pathogenesis of various human diseases

Mosmann first proposed the existence of subsets of CD4+ T cells that produce distinct types of cytokines. Native T lymphocytes (Thp cells) differentiates into either CD4+ Th1 cells that produce IL-2, IFN gamma, and lymphotoxin which promote cell-mediated immunity, or into Th2 cells that produce IL-4, IL-5, IL-6, IL-10 and IL-13, which promote antibody production and humoral immunity. These T cell subsets reciprocally regulate one another since one of the Th1 products, IFN gamma, inhibits the proliferation and functions of Th2 cells, whereas the Th2 products, IL-4 and IL-10, suppress cytokine production by Th1 cells. A distinct Th1/Th2 divergence determine resistance versus susceptibility to diseases such as leishmaniasis and toxoplasmosis in mice. In allergic diseases such as atopic dermatitis and allergic asthma, allergen-specific T cells acquired the Th2 phenotype. These Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13. These cytokines induce eosinophilia and an Ig class switch to IgG4 and IgE. These Th2 cells are responsible for the enhanced production of IgE antibodies. These findings indicate that Th2 cytokines play an important role in the development of allergic diseases. The importance of cell-mediated immunity, particularly donor-anti-host CTL, in mediating acute GVHD suggests that Th1 cytokines may be important in the induction of acute GVHD. To further characterize the roles of Th1 and Th2 cytokines in the development of acute GVHD, analysis of IL-2, IFN gamma, IL-4 and IL-10 cytokine genes was performed by RT-PCR on biopsied skin specimen. An increase in mRNA expression for IL-2 and IFN gamma was observed, whereas there was no significant increase in IL-4 and IL-10 mRNA. These data suggest that Th1 cytokines may be essential for the development of acute GVHD. It is apparent that Th1 cytokines are generally harmful to the maintenance of pregnancy. We have shown that Th2 cytokines are produced by maternal T lymphocytes at the maternal-fetal surface (retroplacental blood lymphocytes). This finding strengthens the hypothesis of a significant contribution of Th2 cytokines to a successful pregnancy.     

Measurement of allergen-specific IGG for diagnosis of atopic diseases in children]

The concentrations of allergen-specific IgE were measured in 146 children with respiratory, skin, and mixed forms of allergic diseases. The spectrum of the allergens was determined and specific features for each of the studied patient populations defined.     

Partial TCR signals delivered by FcR-nonbinding anti-CD3 monoclonal antibodies differentially regulate individual Th subsets

Anti-CD3 mAbs with low FcR affinity prolong graft survival in the absence of the cytokine-mediated toxicity observed with conventional anti-CD3 treatment. Previous studies have shown that FcR-nonbinding anti-CD3 mAbs suppress immune responses, at least in part, by delivering a partial signal resulting in Th1 unresponsiveness. In this study, the biochemical and functional consequences of FcR-nonbinding anti-CD3 treatment for various activated T cell populations were examined. In contrast to Th1 cells, FcR-nonbinding anti-CD3-treated Th2 cells secreted IL-4 and proliferated. Furthermore, Th2 cells cultured with the mAb were not rendered unresponsive. Mixed "Th0" populations responded to FcR-nonbinding anti-CD3 by producing IL-4, and showed a selective decrease in IL-2 production following preculture with the mAb. The stimulation of IL-4-producing cells did not reflect a more complete TCR signal, since similar defects in zeta, ZAP-70, and MAP kinase phosphorylation were observed in Th1 and Th2 cells. Despite the proximal signaling defects, FcR-nonbinding anti-CD3 induced nuclear translocation of NF-ATc. Thus, Abs that deliver partial TCR signals may promote development of a Th2 phenotype during the course of an immune response via selective effects on different Th subsets.     

Interleukin 4, total and allergen-specific immunoglobulin E antibodies in the blood of individuals with an atopic constitution

INTRODUCTION: The recently published data suggest that atopic patients have an enhanced ability to produce IL-4, even in response to antigens other than common environmental allergens or helminthic components. This aberrant IL-4 production by Th cells may be one of the immune alterations encoded by nonMHC genes in the control of basal IgE level [1-7]. The existence of atopen-CD4+ T cell clones that have diverse repertoires might be relevant for aetiopathogenesis of atopic diseases [8-10]. Being subjected continuously to the environmental antigens (allergens), the immune system, especially T cells, is permanently activated and stimulated in response to that challenge. Due to that, substantial amount of diverse cytokines (and their soluble receptors) could be found in the serum. Accordingly, it could be expected to find raised serum IL-4 concentration in atopic persons. In this study we investigated concentrations of IL-4, total IgE and allergen-specific IgE in sera of atopic persons susceptible to pollen allergens. The main goal was to estimate whether the immunologic profile of atopics is defined by increased serum concentration of IL-4 apart from total and allergen-specific serum IgE. METHODS: Patients. We selected 9 atopic patients in the range of 19-35 years, susceptible to grass or weed pollens and suffering from allergic rhinoconjuctivitis. There were 6 females and 3 males (mean age 31.2 years). Atopic allergy was proven by clinical history, skin prick test and by means of in vitro test (positive serum allergen-specific IgE antibodies, RAST/radioallergosorbent tests [11]. As controls we randomly selected B blood donors (6 females, 2 males, mean age 31.3). Study design. The study was carried out from August till September 1995. None of selected patients had previously accomplished allergen-specific immunotherapy nor had been medicated by corticosteroids (topic or systemic) [12-15]. Selected persons were not allowed to take medications such as antihistamines, beta-2 agonists or tricyclic antidepressants at least 10 days prior to the study. We performed in vitro tests for determining concentrations of IL-4, total and pollen-specific serum IgE. Sera were sampled in the morning and were stored frozen at -20 degrees C until analysis. Determination of IL-4, total and allergen specific IgE in serum. Interleukin-4 measurement was carried out in blind fashion with an ELISA kit (Intertest-4 ELISA, Genzyme, Cambridge) according to the manufacturer's instructions. A reference curve was obtained by plotting the IL-4 concentration of several standard dilutions versus an absorbency. The determination limit was 0.045 pg/ml. For determining total serum IgE we used commercially available enzyme immunoassay (EIA Phadezym IgE PRIST, Pharmacia, Uppsala). Normal values was up to 120 IU/L (international unit per ml). Allergen specific IgE was determined by semiquantitative EIA method (RAST Phadezym, Pharmacia, Uppsala) and expressed in Phadebas RAST Unit (PRU) according to standard curve done by manufacturer. Study was carried out after approval of the Ethic Committee of our Hospital and with the obtained patients consents. For statistical analysis we used nonparametric tests (U-test and the Spearman rank correlation). For all comparisons, statistical significance was considered to be present if p < 0.05. RESULTS: Among selected patients, 4 persons had high concentration of serum IgE antibodies (RAST class 4) against grass pollens, two persons had IgE-specific (RAST, class 4) to weed pollens and 2 patients had IgE antibodies (RAST class 4) against weed and grass pollens simultaneously. RAST of class 3 was registered in the serum of one person. Registered serum total IgE, allergen-specifc IgE and IL-4 are shown in Table 1. Serum IL-4 was significantly higher in atopics (U-test, SR 43.53, p.05) as well as total IgE (ER 493, p.05). In atopics, IL-4 was not in correlation with either total or allergen-specific IgE (IL-4 versus total IgE, p = +0.190;     

Effect of pharmacist''s instruction on the treatment of asthmatics with inhaled steroid

BACKGROUND: The clinical efficacy and safety of local nasal immunotherapy (LNIT) with lyophilized 'macronized' powder has been demonstrated. However, the immunological changes possibly induced by LNIT which may account for the clinical improvement are still unclear. OBJECTIVE: To investigate the effects of a successful LNIT-treatment on the allergen-driven T cell response, cytokine secretion and IgE and IgG antibody production. METHODS: Three groups (untreated, subcutaneous immunotherapy- SIT- and LNIT-treated) of grass-sensitive patients suffering from seasonal rhinitic symptoms were ramdomized for the 2-year study. The proliferative response of PBMC to purified Rye-1 allergen and serum levels of grass-specific IgE and IgG were evaluated before treatment and during the 2-year subsequent pollination periods. The proliferative response of allergen-specific short-term T-cell lines, as well as production of allergen-driven cytokine by PBMC, were also assessed. RESULTS: Both SIT and LNIT induced a significant reduction of symptom scores during the pollination season. SIT, but not LNIT, induced a significant change in serum levels of allergen-specific IgE and IgG antibody. By contrast, both SIT and LNIT reduced the increase of the proliferative response of allergen-specific T cells driven by natural allergen exposure and significantly decreased T cell proliferation to low doses of allergen, as shown also by the mitogenic index of allergen-specific T-cell lines. A reduced IL-4 and IFNgamma production by PBMC of LNIT- and SIT-treated patients was also observed in the absence of a clearcut TH2-TH1 switch. CONCLUSIONS: These data suggest that a common mechanism of both LNIT and SIT is the induction of T-cell tolerance, thus providing a rational basis to explain why LNIT may be clinically successful in allergic patients with rhinits.     

Immune response profile in patients with active tuberculosis in a BCG vaccinated area

Tuberculosis patients with pulmonary (N = 95) or lymph node disease (N = 23) were assessed for Th1 responses (PPD skin test and lymphocyte blastogenic and interferon gamma) and Th2 responses (polyclonal and antigen specific IgE). Skin test responses to PPD and lymphocyte proliferative responses to crude mycobacterial antigens (PPD, culture filtrate and sonicate) and recall antigens (tetanus toxoid and streptolysin O) were significantly suppressed (p < 0.001) in patients with pulmonary disease compared to endemic controls. However, mitogen (phytohemagglutinin)-stimulated responses were comparable in patients and controls. Polyclonal and antigen specific (M. tuberculosis culture filtrate) IgE responses which are considered to be surrogate markers for Th2 responses were significantly higher in patients with pulmonary disease compared to healthy endemic controls (Mann Whitney analysis p < 0.01). Patients with lymph node disease showed strong Th1 responses but did not show significant responses for either polyclonal or antigen specific IgE. Thus overall suppression of T cell memory response was observed only in patients with pulmonary disease but not in patients with lymph node disease suggesting that sequestration of antigen in different compartments leads to differential activation of Th1 and Th2 responses. PPD skin test responses were highly positive in endemic controls (47% positive) and household contacts (86% positive). Furthermore, PPD positivity decreased with disease severity. Therefore PPD positivity in a BCG vaccinated TB endemic area cannot be used as a diagnostic marker for active tuberculosis particularly in advanced disease.     

Demonstration of the Th1 to Th2 cytokine shift during the course of HIV-1 infection using cytoplasmic cytokine detection on single cell level by flow cytometry

OBJECTIVE: To characterize changes of Th1/Th2 cytokine production by peripheral blood mononuclear cells (PBMC) that occur during the course of HIV infection by cytoplasmic cytokine staining on single cell level. DESIGN AND METHODS: Mitogen-stimulated PBMC from 16 healthy donors, 18 HIV-1-infected individuals without AIDS and 14 patients with AIDS were stained intracellularly with fluorescein-labelled MAb against interleukin (IL)-2, IL-4, IL-10 and interferon (IFN)-gamma. Additionally, co-staining of CD4+ T-cell, CD8+ T-cell, natural killer (NK) cell, B-cell and monocytic markers was performed. Fluorescence staining was analysed by three-colour flow-cytometry. RESULTS: A reduced percentage of IL-2 and IFN-gamma (Th1 type)-producing cells among CD4+ T cells from HIV-1-infected individuals could be demonstrated. There was a continuous decrease of IFN-gamma-producing CD4+ T cells in the course of HIV infection and a dramatic reduction of IL-2-expressing cells among CD4+ T cells in patients with AIDS. In contrast to Th1 cytokines, the frequency of Th2 cytokine expressing cells among CD4+ T cells increased in HIV-infected individuals. The maximum frequency of IL-4-expressing cells among CD4+ T cells was seen in HIV-infected individuals without AIDS, whereas the rate of IL-10-producing cells was highest in patients with AIDS. In HIV-infected individuals no significant proportion of Th0 cells expressing both Th1 and Th2 cytokines was detectable. In CD8+ T cells the percentage of IL-2 was expressing cells decreased continuously accompanied by a strong increase of the frequency of IFN-gamma-producing cells. CONCLUSION: The decreased percentage of cells expressing IL-2 and IFN-gamma in conjunction with an increased proportion of IL-4- and IL-10-producing cells among the CD4+ T cells in HIV-1-infected individuals demonstrate a Th1 to Th2 cytokine shift in the course of HIV infection on a single cell level. There was no evidence of a Th1 to Th0 cytokine shift. In addition to the loss of CD4+ T cells in HIV infection, the qualitative changes of Th1/Th2 cytokine expression may serve as a marker for progressive failure of cell-mediated immunity.     

Antigen dose-dependent differences in IgE antibody production are not due to polarization towards Th1 and Th2 cell subsets

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL-4 and IL-10 but reduced levels of IL-12. The release of IFN-gamma is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4+ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose-dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.