Demonstration of direct involvement of cytokines in graft-versus-host reactions using an in vitro human skin explant model.

A human skin explant model was used to investigate the role of cytokines in graft-versus-host reactions (GVHR). Responder cells from HLA mismatched mixed lymphocyte cultures (MLC) produced GVHR (Grades I-III) in skin explant assays. Cell-free supernatants from these experiments also induced similar histopathological changes in the skin. The greatest degree of correlation between the GVHR observed with responder cells and the supernatant was shown with CD4 enriched MLC (p less than 0.001). Supernatants were assayed for tumour necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) and CD4 enriched MLC populations produced high levels of these cytokines. These results correlated with the grade of GVHR observed in skin explant assays. The GVHR produced by the supernatant alone could be inhibited by both anti-IFN gamma and anti-TNF alpha polyclonal antibodies. The results suggest that TNF alpha and IFN gamma are directly involved in tissue damage during graft-versus-host disease in allogeneic transplant in man.     

Relationship between IFN-gamma and skin test responsiveness to Mycobacterium tuberculosis PPD in healthy, non-BCG-vaccinated young adults in Northern Malawi.

SETTING: Rural northern Malawi, where vaccination with BCG Glaxo (1077) provides protection against leprosy but not against pulmonary tuberculosis. OBJECTIVE: To evaluate the patterns of responsiveness to purified protein derivative of Mycobacterium tuberculosis (PPD) in terms of delayed type hypersensitivity (DTH) and interferon-gamma (IFN-gamma) production. DESIGN: IFN-gamma was measured in 6 day whole blood cultures diluted 1 in 10, stimulated with PPD RT48, and the results compared to the DTH response to PPD RT23. A total of 633 individuals aged 12 to 28 years, without prior BCG vaccination, were recruited. RESULTS: Overall, 63% of subjects made a positive IFN-gamma response (defined as >62 pg/ml), and 37% gave a DTH induration of >5 mm. A strong correlation between skin test and IFN-gamma responses was observed, although with interesting exceptions: 13/270 individuals with zero DTH showed IFN-gamma responses >500 pg/ml, and 7/53 individuals with >10 mm induration showed IFN-gamma responses < or = 62 pg/ml. The prevalence of skin test responsiveness increased with age, and was higher among older males than females; age-sex patterns were less clear for IFN-gamma production. CONCLUSION: The 6 day IFN-gamma response to PPD correlates well with Mantoux skin test induration. The discordant individuals may represent important subsets in terms of protective immunity and risk of clinical tuberculosis.     

Effects of Th1 and Th2 cytokines on cytokine production and ICAM-1 expression on synovial fibroblasts.

OBJECTIVES--To investigate the influence of the Th1 and Th2 lymphokines interleukins (IL)-4 and IL-13, interferon gamma (IFN gamma), and several monokines on the adhesion of mononuclear cells to synovial fibroblasts and intercellular adhesion molecule-1 (ICAM-1) expression and cytokine production of synovial fibroblasts in patients with osteoarthritis. METHODS--Synovial fibroblasts were isolated from patients with osteoarthritis and stimulated with IL-1 beta, IL-4, IL-6, IL-10, IL-12, IL-13, tumour necrosis factor alpha (TNF alpha), and IFN gamma. Subsequently, we determined the production of IL-1 alpha, IL-1 beta, IL-6, IL-10, IL-12, IFN alpha and TNF alpha, and the expression of ICAM-1 lymphocyte function associated antigen 3 (LFA-3), BB7, and major histocompatibility complex class II molecules on these cells. Furthermore, the adhesion of freshly isolated mononuclear cells from the peripheral blood was tested using a colourimetric cell-cell adhesion assay. RESULTS--Only production of IL-6 and the expression of ICAM-1 were observed. IL-1 beta and TNF alpha were the most potent stimulatory mediators of both cytokine production and ICAM-1 expression. IL-4 and IL-13 had differential effects as they upregulated cytokine production but downregulated IFN gamma induced ICAM-1 expression. In functional adhesion assays, TNF alpha, IL-1 alpha and, to a lesser extent, IFN gamma led to increased adhesion of mononuclear cells, whereas IL-4 and IL-13 had no effect. CONCLUSIONS--Our data indicate that Th1 and Th2 lymphokines can modulate the function (cytokine production and expression of adhesion molecules) of synovial fibroblasts.     

Cytokine responses induced by Mycobacterium tuberculosis in patients with HIV-1 infection and tuberculosis.

OBJECTIVE: Tuberculosis (TB) is an important opportunistic infection in HIV patients. Immune responses to Mycobacterium tuberculosis in HIV/TB patients were evaluated. METHODS: Fifteen patients with HIV/TB, ten with HIV, four with TB, and five controls were enrolled. Peripheral blood mononuclear cells were isolated and stimulated with mycobacterial antigen (PPD). Interferon (IFN)-gamma and TNF-alpha in culture supernatants were measured by ELISA. RESULTS: IFN-gamma and TNF-alpha production after PPD stimulation was markedly decreased in HIV patients, but not in HIV/TB patients. In HIV patients with a CD4 cell count of less than 200/mm3, IFN-gamma and TNF-alpha production after PPD stimulation was higher in HIV/TB patients than in HIV patients. Cytokine responses to M. tuberculosis reconstituted after highly active antiretroviral therapy (HAART) and were prominent in HIV/TB patients. CONCLUSIONS: Cytokine responses to M. tuberculosis were retained in HIV-infected patients with tuberculosis, even in patients with a CD4 cell count of less than 200/mm3, and reconstituted after HAART.     

A 19-year follow-up of tuberculin reactors. Assessment of skin test reactivity and in vitro lymphocyte responses

Tuberculin skin test reactivity decreases with time such that repeat PPD skin testing may result in reactions of less than 10 mm. This reactivity may be boosted in some individuals with a second tuberculin skin test. The immunologic basis of these observations remains unclear. We studied the relationship between skin-test reactivity and in vitro blastogenic response to PPD in a cohort of 22 individuals (aged 28 to 81 years) known to be tuberculin reactors (induration greater than or equal to 10 mm) in 1970. In 1989, 18 subjects remained reactive to PPD on the first skin test and responded to PPD in vitro (mean incorporation of 3H-thymidine by peripheral blood mononuclear cells, 22,650 cpm). Three subjects reverted (induration in response to PPD less than 10 mm) and lost in vitro reactivity to PPD (mean incorporation of 3H-thymidine, 2,205 cpm). One subject boosted (increase of induration of at least 6 mm to greater than or equal to 15 mm) on the second skin test and showed a concomitant in vitro boost in the blastogenic response to PPD (from 1,008 cpm to 47,837 cpm). In this cohort, interpretation of the two-step tuberculin skin test correlated closely with in vitro proliferative responses. Over a 19-year period, the majority of individuals maintained skin test reactivity and strong in vitro responses to PPD despite a lack of ongoing exogenous exposure to Mycobacterium tuberculosis. The immunologic basis for reversion appears to depend in part on a loss of lymphocyte blastogenic capacity. In the one individual who exhibited the booster phenomenon, repeat antigen stimulation resulted in a dramatic increase in the in vitro blastogenic responses.     

Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia

Purified protein derivative (PPD) skin testing is used to identify persons infected with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immune responses to Mtb. However, lack of skin induration to intradermal injection of PPD or PPD anergy is observed in a subset of patients with active tuberculosis (TB). To investigate the sensitivity and persistence of PPD reactivity and its in vitro correlates during active TB disease and after successful chemotherapy, we evaluated the distribution of skin size induration after intradermal injection of PPD among 364 pulmonary TB patients in Cambodia. A subset of 25 pulmonary TB patients who had a positive skin reaction to mumps and/or candida antigens showed persistent anergy to PPD after successful completion of TB therapy. Strikingly, in vitro stimulation of T cells from persistently anergic TB patients with mumps but not PPD resulted in T cell proliferation, and lower levels of IL-2 and IFN-gamma and higher levels of IL-10 were detected in PPD-stimulated cellular cultures from PPD-anergic as compared with PPD-reactive pulmonary TB patients. These results show that anergy to PPD is antigen-specific and persistent in a subset of immunocompetent pulmonary TB patients and is characterized by antigen-specific impaired T cell proliferative responses and a distinct pattern of cytokine production including reduced levels of IL-2.     

Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production.

In tuberculosis, Mycobacterium tuberculosis (MTB)-stimulated T-cell responses are depressed transiently, whereas antibody levels are increased. Lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) from Pakistani tuberculosis (TB) patients to both mycobacterial and candidal antigens were suppressed by approximately 50% when compared to healthy purified protein derivative (PPD)-positive household contacts. Production of interferon gamma (IFN-gamma) in response to PPD also was depressed by 78%. Stimulation with PPD and the 30-kDa alpha antigen of MTB (30-kDa antigen) induced greater secretion of transforming growth factor beta (TGF-beta), but not interleukin 10 (IL-10) or tumor necrosis factor alpha (TNF-alpha), by PBMCs from TB patients compared to healthy contacts. The degree of suppression correlated with the duration of treatment; patients treated for <1 month had significantly lower T-cell blastogenesis and IFN-gamma production and higher levels of TGF-beta than did patients treated for >1 month. Neutralizing antibody to TGF-beta normalized lymphocyte proliferation in response to PPD, partially restored blastogenesis to candidal antigen, and significantly increased PPD-stimulated production of IFN-gamma in TB patients but not in contacts. Neutralizing antibody to IL-10 augmented, but did not normalize, T-cell responses to both PPD and candida in TB patients and candidal antigen in contacts. TGF-beta, produced in response to MTB antigens, therefore plays a prominent role in down-regulating potentially protective host effector mechanisms and looms as an important mediator of immunosuppression in TB.     

Clinico-immunological studies of pulmonary tuberculosis in the elderly

We studied tuberculin reactivity and clinical course after starting chemotherapy in patients with active pulmonary tuberculosis divided by four age-groups less than 29, 30-49, 50-69 and 70 years and more. The skin test to tuberculin purified protein derivative (PPD) was examined in 178 cases of active pulmonary tuberculosis, 120 cases of lung cancer, 25 cases of atypical mycobacteriosis and 466 cases of the other respiratory diseases. The average size of tuberculin reaction in pulmonary tuberculosis decreased with age, but significantly higher than that in patients with other nontuberculous pulmonary diseases of the same age-group. The size of PPD skin test in the group of 70 years and more was significantly lower than other age-groups in pulmonary tuberculosis. We compared the time required for negative conversion of sputum by culture after primary chemotherapy among the different age-groups in pulmonary tuberculosis. It revealed that the time for negative conversion tended to be longer with age, and the time in the group 70 years and more was significantly longer than that of the group less than 29 years of age, although no significant differences in the radiographic severity and conditions of chemotherapy were observed. Finally, the PPD-induced lymphocyte proliferation test in vitro was done in newly diagnosed patients with pulmonary tuberculosis. The patients were divided into two groups by the size of PPD skin test (high responder more than 16 mm and low responder less than 15 mm of erythema induced by PPD).(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of interferon-gamma production in killed BCG-pretreated mice after stimulation with staphylococcal enterotoxin A

Staphylococcal enterotoxin A (SEA), a T cell mitogen, was found to induce a high level of interferon-gamma (IFN-gamma) in mice which had been immunized with killed Bacillus Calmette-Guérin (BCG) in water-in-oil-in-water (W/O/W) emulsion. The phenomenon was analysed by in vivo and in vitro experiments, and the following results were obtained. 1. The SEA-induced IFN was inactivated by treatment with 0.2M glycine-HCl (pH 2.0) but not by heating at 56 degrees C for 30 min. nor by treatment with anti-IFN alpha/beta antibodies, and the fact suggest that the IFN belonged to the gamma type. 2. Treatment of the BCG-sensitized mice with silica and 2-chloroadenosine (2CA), specific lethal agents for macrophages, reduced the SEA-induced IFN production. 3. The SEA-induced IFN production occurred in mice immunized with BCG either intravenously or intraperitoneally, although they showed weak or no footpad reaction to purified protein derivatives (PPD). In contrast, mice sensitized subcutaneously with BCG showed strong foodpad reaction to PPD but not the SEA-induced IFN production. Thus, the mere presence of BCG-sensitized T cells does not appear to result in the SEA-induced IFN production. 4. In vitro experiments, in which SEA-induced IFN production was determined in the culture of BCG-sensitized spleen cells, showed that principal IFN-producing cells were Lyt-1+ T cells. 5. Deprivation of macrophages from BCG-sensitized spleen cell population by passing through Sephadex G-10 column reduced the SEA-induced IFN production in the culture. Addition of 2CA to the culture medium also reduced the SEA-induced IFN production by the BCG-sensitized spleen cells. 6. The SEA-induced IFN production in the culture of the BCG-sensitized spleen cells was suppressed in the presence of lipoxygenase inhibitor, i.e., caffeic acid or nordihydroguaiaretic acid. 7. The plastic adherent spleen cells (i.e. macrophages) from mice sensitized with BCG produced leukotriene C4 (LTC4). The suppression of the SEA-induced IFN production of BCG-sensitized spleen cells in the presence of the lipoxygenase inhibitor was prevented by addition of synthetic LTC4. These results suggest that LTC4 released from macrophages activated by BCG causes production of IFN-gamma by BCG-sensitized T cells responding to SEA.     

The immune response to Mycobacterium tuberculosis in HIV-infected and uninfected adults in Uganda: application of a whole blood cytokine assay in an epidemiological study.

SETTING: Out-patient clinic, Entebbe, Uganda. BACKGROUND: It has been proposed that 'type 1' cytokines are essential in protective immunity to Mycobacterium tuberculosis and that suppression of 'type 1' or a switch to a 'type 2' profile is deleterious. We employed a simple assay to examine whether the dependence of the immunological responses to mycobacterial antigens on a range of explanatory factors could be determined in a population where tuberculosis is endemic. OBJECTIVE: To determine the relationship between the tuberculin skin test response and cytokine profile, and the effect of human immunodeficiency virus (HIV) infection. DESIGN: A cross-sectional study of 97 Ugandan adults (22 HIV-positive, 75 HIV-negative). Whole blood was stimulated in vitro using mycobacterial antigens (purified protein derivative [PPD] and culture filtrate proteins [CFP]). 'Type 1' cytokines (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]), 'type 2' cytokines (IL-5 and IL-10) and tumour necrosis factor alpha (TNF-alpha) were measured in culture supernatants. RESULTS: Among HIV-negative subjects, a positive tuberculin skin test was associated with type 1 or mixed (type 1 + type 2) cytokine production, but a positive IFN-gamma response also occurred in a proportion of tuberculin skin test negative subjects (36% for PPD, 17% for CFP). In association with HIV infection, IFN-gamma responses to mycobacterial antigens were profoundly impaired (odds ratio [OR] 0.10 for PPD, 0.06 for CFP, P< or =0.001), but production of IL-2, IL-5 and TNF-alpha was relatively sustained, and IL-10 increased or sustained (OR 3.97 for PPD, P = 0.01, 1.14 for CFP, P = 0.99). CONCLUSION: The type 1/type 2 cytokine balance was not defined by the tuberculin skin test response, and may have a closer relation to protective immunity. IFN-gamma production was strikingly impaired in association with HIV infection, while production of type 2 cytokines was sustained or increased. Use of a simple assay allowed a large sample of subjects to be examined, producing epidemiologically meaningful results.     

Human in vitro immune responses to Mycobacterium tuberculosis.

SETTING: T helper cells can be divided into 2 subsets on the basis of their cytokine generation. T helper 1 cells secreting gamma interferon and interleukin 2 appear to be more prominent in patients with limited tuberculous disease. OBJECTIVE: The purpose of this study was to evaluate human T helper cell immune responses to mycobacterial antigens in vitro and correlate these with the clinical features of patients with tuberculous infection or disease. DESIGN: We studied 51 subjects and 11 controls who were grouped according to disease involvement as follows: 1) Mantoux negative, BCG negative, no disease; 2) Mantoux positive, no disease; 3) localized extrapulmonary; 4) healed pulmonary; 5) active pulmonary; and 6) miliary/disseminated. Peripheral blood mononuclear cells were cultured with PHA, PPD or Tetanus Toxoid, proliferation assessed and the supernatant analysed using an ELISA for IFN gamma. ELISA was also used to measure M. tuberculosis specific antibodies in the serum. RESULTS: Mantoux size correlated with PPD proliferation r = 0.5, P = 0.005 and gamma IFN production r = 0.36, P < 0.01. All groups produced abundant gamma IFN although there was a trend toward higher production in groups 3 and 4. M. tuberculosis specific IgA (P = 0.003) and IgG1 (P = 0.002) was higher in groups 5 and 6. Those patients with limited disease (groups 2-4) had significantly lower levels of IgG4 than patients with severe disease (groups 5 & 6) (P < 0.02). CONCLUSION: In conclusion patients with healed or extrapulmonary disease have immune responses in vitro suggestive of a TH1 (cell mediated immune) response, whereas patients with miliary/disseminated disease have antibody production suggestive of a TH2 response, together with high gamma IFN production. Both TH1 and TH2 responses may be necessary for host protection if there is a high bacillary load.     

Immunoprofile studies in patients with pulmonary tuberculosis. IV. Tuberculin skin reaction and test of inhibition of blood leukcoyte migration

Study of the relationship existing between in vivo and in vitro correlates of cell immunity has revealed that there is no marked correlation between the magnitude of skin induration of the tuberculin skin test and the degree of inhibition of migration of blood leukocytes stimulated by PPD in patients with newly detected, bacteriologically confirmed pulmonary tuberculosis.     

Two-stage skin testing for tuberculosis in a domiciliary population

We scheduled two-stage skin testing for tuberculosis with Candida and mumps controls on 618 residents of our domiciliary unit at the Veterans Administration Medical Center, Johnson City, Tennessee. Of the 618 residents in the unit, 30 (4.8%) were not available for evaluation, 77 (13%) had a prior history of active tuberculosis or positive skin test, and 1 resident refused testing. Of these 510 patients who received first-step testing with purified protein derivative (PPD), 153 (30%) had greater than to 10 mm induration. Those patients with less than 10 mm induration had a repeat PPD 2 wk later. Fifty-nine (19.2%) of the 307 patients who received a second PPD had a booster response. A total of 50.9% of the residents had evidence of tuberculosis exposure by skin testing. There were no differences between patients with significant and nonsignificant reactions when comparing age, length of stay, functional status evaluated by Karnofsky scale, or number of underlying diseases. Second test conversion occurred in 4.3% of those patients who had been in the unit for less than 1 month and in 36% of those who had been residents for a period of 3 to 6 months (p less than 0.05). Regardless of the size of the initial reading, it is important to perform a two-stage PPD in residents of chronic care facilities who have a negative first test. INH prophylaxis should be considered in patients admitted to chronic care facilities such as the domiciliary when they have significant Mantoux reactions.     

Mucosal tumour necrosis factor alpha and interleukin-6 in patients with Helicobacter pylori associated gastritis.

The production of tumour necrosis factor alpha (TNF alpha) and interleukin-6 by human antral mucosa during short term culture in vitro has been measured by enzyme linked immunosorbent assay. TNF alpha and interleukin-6 concentrations in culture supernatants were significantly greater (p less than 0.001) in patients infected with Helicobacter pylori, all of whom had chronic gastritis, than in patients who were H pylori negative with histologically normal gastric mucosa. Among H pylori colonised patients, TNF alpha concentrations were significantly higher in those with active gastritis and neutrophil infiltration into the epithelium than in those with inactive gastritis. In contrast, interleukin-6 concentrations were raised in both active and inactive gastritis. This study shows that H pylori gastritis is associated with increased gastric mucosal production of TNF alpha and interleukin-6 and that the nature of the mucosal cytokine response varies with the immunohistology of the disease. Inflammatory cytokines generated locally within the gastric mucosa could be relevant to the gastric physiology of H pylori infection.     

Prognostic values of tumor necrosis factor/cachectin, interleukin-1, interferon-alpha, and interferon-gamma in the serum of patients with septic shock. Swiss-Dutch J5 Immunoglobulin Study Group

Serum concentrations of immunoreactive tumor necrosis factor/cachectin (TNF), interleukin-1 beta (IL-1 beta), interferon-gamma (IFN gamma), and interferon-alpha (IFN alpha) were prospectively measured in 70 patients with septic shock to determine their evolution and prognostic values. In a univariate analysis, levels of TNF (P = .002) and IL-1 beta (P = .05) were associated with the patient's outcome, but not IFN alpha (P = .15) and IFN gamma (P = .26). In contrast, in a stepwise logistic regression analysis, the severity of the underlying disease (P = .01), the age of the patient (P = .02), the documentation of infection (nonbacteremic infections vs. bacteremias, P = .03), the urine output (P = .04), and the arterial pH (P = .05) contributed more significantly to prediction of patient outcome than the serum levels of TNF (P = .07). After 10 days, the median concentration of TNF was undetectable (less than 100 pg/ml) in the survivors, whereas it remained elevated (305 pg/ml, P = .002) in the nonsurvivors. Thus, in patients with septic shock due to various gram-negative bacteria, other parameters than the absolute serum concentration of immunoreactive TNF contributed significantly to the prediction of outcome.     

Down-regulation of the nonspecific and antigen-specific T cell cytokine response in ankylosing spondylitis during treatment with infliximab

OBJECTIVE: Treatment of active ankylosing spondylitis (AS) with the monoclonal tumor necrosis factor alpha (TNF alpha) antibody infliximab is highly clinically effective. This study was undertaken to investigate the precise mechanism of action of anti-TNF alpha treatment in AS. METHODS: Cytokine expression of CD4+ and CD8+ T cells was investigated before and 6 and 12 weeks after the start of treatment in 10 patients treated with infliximab, and before and after 6 weeks of treatment and 6 weeks after placebo was switched to infliximab in 10 patients treated initially with placebo. Peripheral blood mononuclear cells (PBMCs) were stimulated for 6 hours either nonspecifically with phorbol myristate acetate (PMA)/ionomycin or antigen specifically with a pool of 46 overlapping 18-mer peptides derived from the G1 domain of aggrecan. Cells were stained for T cell surface markers CD4 and CD8 and for the intracellular cytokines interferon-gamma (IFN gamma), TNF alpha, interleukin-4 (IL-4), and IL-10. Positive cells were quantified by flow cytometry. For monocyte-derived cytokines, PBMCs were stimulated with lipopolysaccharide (LPS) for 18 hours and TNF alpha and IL-10 in the supernatant were measured by enzyme-linked immunosorbent assay. RESULTS: Compared with baseline, infliximab treatment induced a significant decrease at 12 weeks in the number of CD4+ and CD8+ T cells that were positive for IFN gamma and TNF alpha upon PMA/ionomycin stimulation (P = 0.005). A significant reduction had already begun to occur at 6 weeks. No change in the percent IFN gamma or TNF alpha positivity among CD4+ and CD8+ subpopulations was observed after 6 weeks in patients treated with placebo. However, when these patients began infliximab treatment after 6 weeks of receiving placebo, there was a similar significant decrease in IFN gamma and TNF alpha production by CD4+ and CD8+ T cells (P < 0.05). Furthermore, infliximab treatment induced a significant reduction in the number of IFN gamma+ and TNF alpha+ CD8+ T cells (P = 0.005 at week 6 and week 12) after antigen-specific in vitro stimulation with G1-derived peptides. Between-group analysis showed that the change in the expression of IFN gamma and TNF alpha in both CD4+ and CD8+ T cells was significantly different between the infliximab and placebo groups (P = 0.001 for all variables). There was no change in the number of IL-10+ or IL-4+ T cells during treatment. No significant change in the production of TNFalpha and IL-10 upon in vitro stimulation of PBMCs with LPS was detectable during infliximab treatment. CONCLUSION: Infliximab down-regulates both IFN gamma and TNF alpha secreted by T cells but does not induce a change in cytokines produced by monocytes during 3 months of treatment. This is likely to be a relevant mechanism for the clinical efficacy of this therapy.     

Inhibitory effects of tumor necrosis factor-alpha and interferon-gamma on deoxyribonucleic acid and collagen synthesis by rat osteosarcoma cells (ROS 17/2.8)

Tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) have potent effects on bone resorption and collagen synthesis in cultured rat long bones. Since the effects of TNF alpha and IFN gamma may result from interaction with multiple cell types, we studied the effects of these cytokines on the synthesis of DNA and collagen in one cell type with osteoblast phenotype, cloned rat osteosarcoma cells (ROS 17/2.8). Recombinant human TNF alpha did not affect DNA synthesis after 48 h with concentrations of 10(-11)-10(-8) M and inhibited DNA synthesis slightly at 10(-6) M. Recombinant rat IFN gamma (5-500 U/ml) caused a dose-dependent inhibition of DNA synthesis. Coincubation with TNF alpha and IFN gamma inhibited DNA synthesis more than maximal doses of either cytokine alone. This enhanced inhibitory effect was due to the induction of a response to TNF alpha by IFN gamma, since preexposure of cells to IFN gamma for 24 h, followed by incubation with TNF alpha alone for an additional 48 h, also resulted in increased inhibition of DNA synthesis. Preexposure to TNF alpha for 24 h, followed by IFN gamma alone, did not increase the inhibition of DNA synthesis. Incubation with either IFN gamma (5-500 U/ml) or TNF alpha (10(-10)-10(-6) M) inhibited the incorporation of [3H]proline into collagen. Coincubation with intermediate concentrations of both cytokines resulted in an inhibitory effect greater than that produced by maximal concentrations of either alone. The results indicate that 1) IFN gamma and TNF alpha have direct actions on osteoblast-like cells in vitro; 2) IFN gamma modulates the DNA response to TNF alpha; and 3) the greater responses to combined cytokines than to high doses of either alone suggest that these cytokines act, at least in part, through different pathways.     

Gamma-Interferon and Soluble Interleukin 2 Receptor in Tuberculous Pleural Effusion

To analysis the difference between systemic and local pleural T cell response in pulmonary tuberculosis, we analyzed interferon (IFN)-gamma and soluble interleukin-2 receptor (sIL-2R) in peripheral blood mononuclear cells (PBMC) culture supernatants and in pleural effusion (PE). We also investigated the association of pleural INF-gamma and sIL-2R levels with development of residual pleural thickening (RPT). The subjects in this study included patients with active pulmonary tuberculosis with or without PE (n = 46), those with nontuberculous PE (n = 32), and healthy tuberculin reactors (n = 20). Measurement of IFN-gamma and sIL-2R were made by ELISA. In pulmonary tuberculosis, IFN-gamma and sIL-2R concentrations in PBMC culture supernatants were lower than those of healthy tuberculin reactors (IFN-gamma; 258.4 +/-111.5 pg/mL versus 2792.5 +/-633.2 pg/mL, sIL-2R; 1465.0 +/-144.4 pg/mL versus 4777.1 +/-178.5 pg/mL, p < 0.05), whereas IFN-gamma and sIL-2R concentrations in PE were higher than those from nontuberculous pleural effusion (IFN-gamma; 1154.4 +/-252.4 pg/mL versus 292.0 +/-68.9 pg/mL, sIL-2R; 9805.2 +/-978.9 pg/mL versus 3426.7 +/-695.6 g/mL, p < 0.05). IFN-gamma and sIL-2R in PBMC culture supernatants were significantly lower in tuberculat patients with PE than those without PE, and the patients with a high value of IFN-gamma or sIL-2R in PE showed a low value of IFN-gamma or sIL-2R in PBMC culture supernatant, respectively. Patients with RPT had significantly higher IFN-gamma and sIL-2R values in their PE compared with those without RPT. These findings suggest that diminished systemic Th1 response in tuberculosis results from the accumulation of activated Th1 cell to the disease site, and that levels of IFN-gamma and sIL-2R in PE are useful posttreatment markers of RPT.     

Flow-cytometric assessment of lymphocyte cytokine production in tuberculosis

We assessed by flow-cytometry the Th1/Th2 profiles in peripheral blood lymphocytes (PBL) from patients with active tuberculosis (TB), before and after antituberculous therapy, and from healthy tuberculin-positive and -negative reactors. PBL from patients showed a reduced potential for Th1-cytokine (notably IFN- gamma) production after culture with a policlonal stimulus. When these PBL from patients were cultured with a M. tuberculosis (MTB)-specific antigen such as PPD (10 microg/ml), there was no detectable production of Th1 cytokines. Only the Th2 cytokine IL10 was detected in PBL from patients but not from controls. However, at the site of the tuberculosis disease, T lymphocytes from bronchoalveolar lavage, after culture with PPD, produced IFN- gamma. After completion of tuberculosis therapy, PBL did not produce IL10. These data indicate that the immunosuppression observed in PBL during active tuberculosis infection may be related to IL10 production, and to the compartmentalization of the antigen-Th1 response to sites of active MTB infection.     

Interferon gamma encapsulated into liposomes enhances the activity of monocytes and natural killer cells and has antiproliferative effects on tumor cells in vitro

Summary The effects of human interferon gamma (IFN gamma) encapsulated into liposomes were investigated in vitro. Monocytes were induced to release a cytotoxic factor with either IFN gamma encapsulated into liposomes, free IFN gamma or lipopolysaccharide (LPS). If IFN gamma was applied in the liposomal form, less IFN activity was required to stimulate monocytes. Most of the cytotoxic factor was secreted during the first 4 h of stimulation. The cytotoxic factor in supernatants from PMNLs was completely neutralized by a monospecific polyclonal antiserum to tumor necrosis factor (TNF). Combining subthreshold doses of IFN gamma liposomes or IFN gamma with lipopolysaccharide synergistically enhanced the release of TNF. In fluorescence analysis, altered expression of the class II HLA-DR antigen on LeuM3 positive monocytes was induced with IFN gamma liposomes as well as with IFN gamma. Not only monocytes but also natural killer (NK) cells were stimulated to higher cytotoxicity by IFN gamma liposomes in a dose-dependent manner. In comparison with IFN gamma, the same amount of activity was necessary for adequate stimulation of NK-cells against the K562 target cells. Furthermore, the antiproliferative effects of IFN gamma liposomes and free IFN gamma on several human tumor cell lines was compared. Among several cell lines tested, U937 and A549 turned out to be sensitive to IFN gamma, and both cell lines reacted with 50% growth inhibition at a lower amount of gamma presented by liposomes than in the free form. These data show production of IFN gamma liposomes which possess immunmodulatory and antiproliferative activity in vitro. In several of the test systems studied, liposome-encapsulated IFN gamma was more effective than free IFN gamma.