Production of cytokines during interaction of peripheral blood mononuclear cells with autologous ovarian cancer cells or benign ovarian tumour cells.

Cytokines produced by tumour and immune cells may play a significant role in a modulation of immune cells response against tumour. We investigated an ability of peripheral blood mononuclear cells (PBMC) of patients with early and advanced stages of ovarian cancer and from non-cancer patients to produce various cytokines in the presence or absence of autologous ovarian cancer (OC) cells or benign ovarian tumour (BOT) cells. Activated PBMC of patients with advanced stage of cancer produced slight amount of interferon gamma (IFN-gamma) and what's more, the production of IFN-gamma was decreased in the presence of OC cells. PBMC of patients with ovarian cancer or benign ovarian tumour generated comparable amounts of interleukin 6 and 10 (IL-6, IL-10), and transforming growth factor beta1 (TGF-beta1). PBMC of the patients with cancer produced higher amount of tumour necrosis factor alpha (TNF-alpha) than PBMC of non-cancer patients. We demonstrated here that the reciprocal contact of OC cells from advanced cancer with autologous PBMC altered the direction of produced cytokines and leads to the down-regulation of IFN-gamma and TNF-alpha as well as to up-regulation of immunosuppressive (IL-10, TGF-beta1) and pro-inflammatory (IL-6) cytokines production.

Frequencies of killer immunoglobulin-like receptor genotypes influence susceptibility to spontaneous abortion.

Natural killer (NK) cells are the most abundant lymphocyte population in the decidua. These cells express killer immunoglobulin-like receptors (KIRs), which upon recognition of HLA class I molecules on trophoblasts may either stimulate NK cells (activating KIRs) or inhibit them (inhibitory KIRs) to produce soluble factors necessary for the maintenance of pregnancy. KIR genes exhibit extensive haplotype polymorphism; individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes. This polymorphism affects NK cell reactivity and susceptibility to diseases, including gynecological disorders. Therefore we KIR-genotyped 149 spontaneously aborting women and 117 control multiparae (at least 2 healthy-born children). Several genotypes (i.e. combinations of various KIR genes) were differently distributed among the patients and control subjects. Differences were observed in the numbers and the ratios of activating to inhibitory KIRs between patients and healthy women: (i) genotypes containing 6 activating KIR genes were less frequent and those containing 6 inhibitory KIR genes were more frequent in patients than in control subjects, and (ii) an excess of inhibitory KIRs (activating-to-inhibitory KIR gene ratios of 0.33 to 0.83) was associated with miscarriage, whereas ratios close to equilibrium (0.86-1.25) seemed to be protective. In addition, the results suggest for the first time that sporadic and recurrent spontaneous abortions as well as miscarriage in the presence or absence of autoantibodies may have different KIR genotypic backgrounds.

The effect of short-term myocardial ischemia on the expression of adhesion molecules and the oxidative burst of coronary sinus blood neutrophils.

We examined the influence of transient myocardial ischemia on the number and function of neutrophils in patients with effort angina (EA). We tested fluorometrically the expression of neutrophil membrane molecules (CD11b, CD11c, CD18) and neutrophil oxidative burst using a chemiluminescence (CL) generation system. The estimations were conducted before, 1 min after and 20 min after percutaneous transluminal coronary angioplasty (PTCA) in 15 patients qualified for the treatment because of single-vessel disease. Eight EA patients subjected to coronary arteriography (CA) comprised a control group. We did not observe any marked changes in leucocytosis or lymphocyte number in peripheral blood (PB) or in coronary sinus blood (CSB) after the procedure. The percentage of granulocytes in coronary blood decreased significantly 20 min after reperfusion. No significant changes in white blood cell count were noted in peripheral blood of PTCA patients or in control CA subjects. Oxidative burst of nonstimulated and fMLP, PMA and zymosan stimulated sinus blood neutrophils was significantly depressed 1 min after inflation, and enhanced 20 min after reperfusion. We found a significant increase in the percentage of the CD11c+ neutrophils from 56.7 +/- 7.4% to 64 +/- 6.5% 20 min after inflation and postischemic decrease in the CD11c molecule expression on CSB neutrophils. Significant positive linear correlation (Rval = 0.71) between inflation time and the CD11c molecule expression on CSB immediately after reperfusion was also noted. The results may reflect local activation of neutrophils in ischemic myocardium as a response to ischemia induced increase of activating stimuli.

The effect of lysosome factors derived from PMNL of multiple sclerosis and systemic lupus erythematosus patients on suppressor cell activity in vitro.

The lysosomal factors obtained from PMNL of healthy persons and patients with multiple sclerosis, systemic lupus erythematosus and other neurological diseases inhibited in vitro the generation of Con A-induced suppressor cell activity. The results obtained proved that these factors from granulocytes of MS-relapse and active LE have a stronger inhibitory effect on suppressor cell activity than on healthy ones.

Protease inhibitors diminish lymphocyte stimulation in vitro.

Lymphocyte stimulation initiates activation of signal transduction pathways presumably enzymatic in nature. The next step includes gene activation and specific protein molecule production. Proteases of different specificity are crucial in enzyme activation: protein cleavage and biologically active molecule production. To elucidate the role of proteases in peripheral blood mononuclear cell (PBMC) activation, the broad specificity inhibitors of thiol (PHMB) and serine (TLCK) proteases have been used in vitro. Both inhibitors diminished the PHA-induced lymphocyte stimulation when applied at the beginning of culture; inhibitory effect was abrogated when inhibitors were introduced to the culture system 4 h later. Exogenous IL-2 abolished inhibition. PHMB activity was reversible whereas TLCK was irreversible. The inhibition of T lymphocyte-enriched proliferation is more distinctive as compared to that of PBMC. It can be concluded that proteases are involved in early events of lymphocyte proliferation and IL-2 production which is responsible for different stages of cell growth.

Modulation of immune response in vitro and in vivo by human granulocyte factors.

The adherence of granulocytes induces secretion of specific granule contents. The secreted proteins were termed granulocyte factors (GF). The experiments in vivo provide evidence that GF play an essential role in the stimulation of PFC in BALB/c mice immunized with SRBC when applied before challenge three times (5 micrograms per mouse), but 50 micrograms per mouse given in the same way diminishes the response. To elucidate this discrepancy, the effect of GF on the generation of suppressor cells (SC) and helper cells (HC) in vitro has been investigated. Antigen specific nonadherent SC or HC were induced in vitro using CBA mice spleen cells incubated with 100 micrograms/ml or 0.1 mg/ml of TNP-KLH, respectively, for 4 days. GF in concentrations of 0.1 to 1 microgram/ml abolish antigen specific SC generation. SC and HC activity was tested in cooperative cultures. Antigen specific SC in delayed hypersensitivity (DTH) to BCG were induced in an in vitro system as above using normal BALB/c spleen cells and 100 micrograms/ml PPD. Nonadherent suppressor cells were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer. DTH was measured by foot-pad reaction. This reaction was positive to PPD in CY treated mice immunized to BCG, while it was suppressed by the transfer of in vitro induced SC. When the SC were induced in the presence of 1 microgram/ml GF, the suppression was abrogated. The higher GF concentrations stimulated SC activities when they were measured in response to a nonrelated antigen and in specific anti-PPD response, but the HC inhibition could not be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cimetidine treatment on suppressor T cells in duodenal ulcer patients.

Changes in the suppressor T-lymphocyte activity were studied in 11 patients with duodenal ulcer during treatment with cimetidine. The drug was administered intravenously in a dose of 200 mg four times a day for a fortnight. Suppressor T-cell activity was determined by the Shou et al. method using two-stage culture before treatment, after 4 days of the treatment, just before drug withdrawal, and 2 days and 2 wk after the treatment. Suppressor T-cell activity significantly decreased soon after starting the treatment, remained low throughout the treatment, and rapidly and significantly increased following drug withdrawal.

Changes in the interleukin-1 and interleukin-2 generation in duodenal ulcer patients during cimetidine treatment.

The effect of cimetidine treatment on the generation of interleukin-1 (IL-1) and interleukin-2 (IL-2) was studied in 11 duodenal ulcer patients. The results obtained were compared with those for untreated healthy subjects. The drug was administered intravenously in a dose of 200 mg four times a day for 8 days. The investigations were performed before, during and 1 wk after cimetidine therapy. IL-1 generation was determined by the ability of supernatants from 2-day cultured adherent cells stimulated by lipopolysaccharide to enhance proliferation of PHA-stimulated mice thymocytes. IL-2 generation was determined by the ability of supernatants from 2-day cultured, PHA-stimulated mononuclear cells to proliferate autologous 17-day cultured T cells. In all ulcer patients IL-1 generation diminished during cimetidine treatment (P less than 0.005). It continued to decrease in 4 subjects and increased in the other 7 ones following drug withdrawal. All the values were higher than those in healthy controls. IL-2 activity in ulcer patients was similar to that in healthy subjects and it increased significantly in all ulcer patients following the onset of the treatment (P less than 0.005) and decreased nearly to the initial values 1 wk after termination of the treatment (P less than 0.005). The present studies indicate that cimetidine, a selective histamine H2-receptor antagonist, deeply changes mechanisms of immunoregulation in patients with duodenal peptic ulcer.

Modification of the neutrophil Fc receptor by neutrophil granule products: its significance for phagocytosis and bactericidal activity.

Preincubation of neutrophils with various amounts of autologous neutrophil granule products induced a dose-dependent decrease in neutrophil Fc receptor expression. However, neutrophil granule products did not affect the neutrophil phagocytic and bactericidal activities.

Effect of pre-S1 antigen on human lymphocyte proliferative responses.

The peripheral blood mononuclear cells (PBMCs) from patients acutely infected with HBV and recovered completely (n = 20), patients with chronic hepatitis B infection (CHB)- (n = 10) and HBsAg-positive carriers (n = 9) and healthy individuals (n = 8) were studied for their in vitro proliferative response to a synthetic pre-S1(20-49)x4 antigen. PBMCs from convalescents showed significant proliferative response in the presence of synthetic pre-S1 antigen. PBMCs from CHB- and HBsAg-positive exhibited reduced proliferative response not only to Pre-S1 antigen but also to nonspecific mitogens. This study suggests that the immune recognition of pre-S1 antigen and response of PBMCs to the pre-S1 antigen may be an important part of the normal human response to HBV infection. Failure to clear the HBV infection with development of the chronic carrier state may be caused by the lack of an efficient pre-S1 antigen-specific response.

The role of receptors for tumour necrosis factor-alpha in the induction of human polymorphonuclear neutrophil chemiluminescence.

Tumour necrosis factor-alpha (TNF-alpha) is a potent mediator of inflammation, which exerts profound effects on polymorphonuclear neutrophils (PMN). TNF-alpha binds to distinct cell surface receptors termed p55 and p75, expressed in approximately equal amounts on the PMN surface. We have studied the effects of TNF-alpha on the priming of F-Met-Leu-Phe (FMLP)-stimulated oxidative metabolism of PMN, using a luminol-enhanced chemiluminescence assay, and have examined the relative roles of PMN receptors for TNF-alpha in priming this oxidative metabolism, using antibodies with p55 and p75 receptor-specific agonistic and antagonistic activities. We have obtained the following results: (1) Antibody Htr-9 with agonistic activity at the p55 receptor mimicked the effect of TNF-alpha; however, a combination of Htr-9 and TNF-alpha did not results in any further increase in chemiluminescence relative to the response observed with TNF-alpha alone. The p75 agonistic antibody MR2-1 actually decreased basal and FMLP-enhanced chemiluminescence. Additionally, MR2-1 substantially inhibited the effects of both TNF-alpha itself and of the p55 agonist Htr-9. (2) Addition of antibodies with antagonistic activities at the p55 (antibody TBP-2) and p75 (antibody Utr-1) receptors resulted in a marked inhibition of the PMN response to TNF-alpha. A combination of both Utr-1 and TBP-2 was most effective at inhibiting the action of TNF. We have confirmed previously published observations that TNF-alpha alone effectively stimulates the oxidative metabolism of PMN in vitro, and that pre-incubation of PMN with TNF-alpha enhances subsequent generation of oxidative metabolites in response to FMLP. We conclude that both p55 and p75 receptors play a critical role in mediating the activation of PMN by TNF-alpha.

Increased expression of neutral endopeptidase (NEP) and aminopeptidase N (APN) on peripheral blood mononuclear cells in patients with multiple sclerosis.

We studied CD10 Ag of neutral endopeptidase (NEP) and CD13 Ag of aminopeptidase N (APN) expression on peripheral blood mononuclear cells (PBMC) as cells activation markers and for their transendothelial migration properties in three groups of MS patients (total 58); with acute exacerbation of MS (n = 18), primary progressive MS (n = 17) and with MS remission (n = 23). The control group (OND) consisted of 24 patients, suffering from other noninflammatory neurological diseases. CD10 Ag and CD13 Ag expression on PBMC was higher in clinically active MS (acute exacerbation and progressive MS) compared to MS remission and OND groups. Our study suggests that CD10 Ag and CD13 Ag can be useful mononuclear cell activation markers in the course of MS. CD13 Ag expression on PBMC may be also the sensitive marker of these cells transendothelial migration properties.

Priming effect of met-enkephalin and beta-endorphin on chemiluminescence, chemotaxis and CD11b molecule expression on human neutrophils in vitro.

The opioid peptides are widely distributed throughout the body, and they are generated during stress and inflammatory reaction. Opioids are involved in the communication between the immune and neuroendocrine systems. In the present study we have investigated the ability of both met-enkephalin and beta-endorphin to stimulate and prime the human neutrophils for enhanced chemiluminescence (CL) and chemotaxis induced with fMLP, OZ or PMA. We have also tested the effect of beta-endorphin and met-enkephalin on CD11a, CD11b, CD18 and CD16 molecule expression on PMN in vitro. PMN from ten healthy donors were incubated in vitro with different concentrations of beta-endorphin or met-enkephalin, and the CL response was evaluated with luminometer. To assess the effect of opioid peptides on CD11a, CD11b, CD18 and CD16 molecule expression the whole blood samples were incubated with different concentrations of the opioids, then the white cells were labelled with respective PE-conjugated MoAb and evaluated by flow cytometry. We have shown that: (1) met-enkephalin and beta-endorphin at physiological concentrations relevant to that of in vivo (10(-8) and 10(-6) M) enhanced fMLP, PMA or OZ stimulated chemiluminescence and induced chemotactic response, (2) High concentrations of beta-endorphin (10(-3) M) or met-enkephalin (10(-5) M) decreased the CL response of PMN in vitro, (3) The opioid peptides at lower concentrations resulted in CD11b and CD18 molecule up-regulation on neutrophils. We may conclude that opioid peptides in physiological concentration are involved in neutrophil priming whereas in higher concentration exert immunosuppressive potency. Opioid peptides like inflammatory cytokines may prime the neutrophils inflammatory response.

Activated T lymphocytes from patients with high risk of type I diabetes mellitus have different ability to produce interferon-gamma, interleukin-6 and interleukin-10 and undergo anti-CD95 induced apoptosis after insulin stimulation.

Type I Diabetes mellitus (DM1) is the effect of T cell dependent autoimmune destruction of insulin producing beta cells in the pancreas islet. T cells are activated in response to islet dominant autoantigens, the result being the development of DM1. Insulin is one of the islet autoantigens responsible for activation of T lymphocyte functions, inflammatory cytokine production and development of DM1. The experiments reported in this study have shown the spontaneous increase of CD95 molecule expression on lymphocytes of the first-degree relatives of DM1 patients. The autoantigen insulin is responsible for stimulation in vitro of potentially hazardous 'memory' lymphocytes to produce interleukin-6 (IL-6) and interleukin-10 (IL-10) interleukins. Insulin induced stimulation of lymphocytes in vitro was observed in patients at high risk of developing diabetes mellitus (prediabetics). Phytohaemagglutinin (PHA) stimulates lymphocytes of all groups in the same way. Stimulated lymphocytes in second cultures undergo apoptosis induced with anti-Fas specific antibodies. The deletion in vitro of resting peripheral lymphocytes is nonfunctional. Insulin activated T lymphocytes, which undergo apoptosis were not observed in peripheral blood of healthy people and in patients with DM1. This observation suggests that insulin is involved as autoantigen in DM1 progression in patients with high risk of diabetes type I. The autoreactive T lymphocytes may persist in peripheral blood of patients with high risk DM1. Defective elimination of autoreactive T cells may result in autodestructive damage of islets beta cells in the prediabetic stage and disease progression to DM1.

The inhibitory effect of granulocyte factor in mixed lymphocyte reaction.

The polymorphonuclear leukocytes (PMNLs) depress mixed leukocyte reaction (MLR) in vitro. The active factor involved in inhibition was found to be localized in the specific granules of PMNL. The adherent granulocytes secreted granulocyte factor (GF) which also diminished MLR. The functional activity of GF is reflected by inhibition of responding cell proliferation exclusively during induction of MLR; moreover, GF depresses generation of the specific suppressor cells associated with MLR. The effect of GF is modified using specific antisera.

Interleukin-1 and interleukin-2 production by peripheral blood mononuclear cells in multiple sclerosis patients.

The production of interleukin-1 (IL-1) and interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBM) was assessed in multiple sclerosis (MS) patients in relapse, chronic progressive MS patients, patients with other neurological diseases (OND) and healthy subjects. Production was defined as the level of IL-1 and IL-2 in PBM supernatants. Neither spontaneous nor LPS-induced IL-1 production differed significantly in MS, OND patients or healthy individuals. On the other hand PHA-induced PBM IL-2 production was significantly less in MS patients in relapse (130 +/- 10.0 U/ml) than in chronic progressive MS patients (172 +/- 9.8 U/ml), OND patients (192 +/- 11.5 U/ml) and healthy subjects (215 +/- 13.8 U/ml) (P less than 0.02). Spontaneous IL-2 production was also diminished in MS patients in relapse (31 +/- 7.2 U/ml) as compared to chronic progressive MS patients (46 +/- 8.8 U/ml) and healthy subjects (49 +/- 11.1 U/ml) (P less than 0.01). Anti-Tac monoclonal antibody was used to study IL-2 receptor expression on the same sample of PBM that was used for IL-2 study. MS patients in relapse had significantly higher levels of IL-2 receptor-positive unstimulated PBM (6.0 +/- 2.2%) as compared to chronic progressive MS (2.0 +/- 0.9%), OND (2.5 +/- 1.1%) and healthy subjects (1.5 +/- 0.7%) (P less than 0.002). We postulate that reduced apparent IL-2 production by PBM of MS patients in relapse may result from immediate IL-2 binding to receptor expressed on activated T lymphocytes and internalization of IL-2-receptor complex.

Granulocyte factor and graft-versus-host reaction in rats. I. The inhibitory effect of granulocyte factor on the local graft-versus-host reaction in rats.

Recent data have proved that PMNLs regulate the immune responses in vitro and in vivo. PMNL specific granules contain an immunoregulatory factor termed the granulocyte factor (GF). The glycogen-induced PMNL infiltration to peritoneal cavity of rats significantly diminished the local GvHR. GF, injected subcutaneously three times at a day of challenge, one and two days after, significantly diminished local GvHR as well. GF treatment before parental lymphocyte injection had no effect on the local GvHR in rats. Inactivation of GF using anti-GF IgG antibodies, abolished the inhibitory effect of PMNLs in GvHR.

The mitogenic and enzymatic activities of polymorphonuclear leukocyte (PMNL) lysosomal proteins examined in vitro.

The lysosomal granules of human and animal PMNL contain the protein factors which stimulate the lymphocyte transformation in vitro. The performed experiments showed that mitogenic activity is not exclusively associated with proteolytic activity. Using column chromatography fractionation, it has been shown that the mitogenic and proteolytic proteins are localized in different fractions. The irreversible inhibition of proteases by di-isopropyl-phosphofluoridate (DFP) increase the mitogenic activity of PMNL lysosomal proteins. The mitogenic proteins are localized in lysosomal granules of secondary density and they are loosely bound to membranes of lysosomal granules.

The role of autoaggressive reactions in recurrent uveitis.

An experimental study in rabbits and clinical study in patients with uveitis was carried out. Experimental uveitis was induced in rabbits sensitized with albumin and antigen was injected intravenously or into the vitrous body of their eyes. Blastic transformation of lymphocytes stimulated with PHA, albumin and extracts of the uvea and cytotoxicity of the serum against normal white blood cells in presence of antigen were determined. In recurrent uveitis blastic transformation induced with uvea antigens, hemagglutinin titers against these antigens and serum cytotoxicity increased. The results indicate a significant role of autoaggression in the pathogenesis of some recurrent eye uveitis cases.

Effect of neutrophils on the growth of human pre-S1-reactive T-cells in vitro.

Granulocyte factor (GF) derives from the specific granules of polymorphonuclear neutrophils. GF possesses an immunoregulatory activity and augments the immune response to antigens in vitro and in vivo. The Pre-S1 sensitive T-cells reactive to Pre-S1 protein in vitro were developed from peripheral blood mononuclear cells of hepatitis B convalescent by in vitro Pre-S1 protein stimulation. The GF involvement in the activation and development of the Pre-S1 specific regulatory T-cells in vitro was studied.