Search

It is widely accepted that oxidized low-density lipoproteins and local infections or endotoxins in circulation contribute to chronic inflammatory process at all stages of atherosclerosis. The hallmark cells of atherosclerotic lesions—monocytes and macrophages—are able to detect and integrate complex signals derived from lipoproteins and pathogens, and respond with a spectrum of immunoregulatory cytokines. In this study, we show strong inhibitory effect of oxLDLs on anti-inflammatory interleukin-10 production by monocytes responding to TLR2 and TLR4 ligands. In contrast, pro-inflammatory tumor necrosis factor secretion was even slightly increased, when stimulated with lipopolysaccharide from Porphyromonas gingivalis—an oral pathogen associated with atherosclerosis. The oxLDLs modulatory activity may be explained by altered recognition of pathogen-associated molecular patterns, which involves serum proteins, particularly vitronectin. We also suggest an interaction between vitronectin receptor, CD11b, and TLR2. The presented data support a novel pathway for pathogen-accelerated atherosclerosis, which relies on oxidized low-density lipoprotein-mediated modulation of anti-inflammatory response to TLR ligands. Electronic supplementary material The online version of this article (doi:10.1007/s10753-012-9472-3) contains supplementary material, which is available to authorized users.

Renal insufficiency predisposes to coronary artery disease (CAD), but also CAD and traditional risk factors accelerate renal function loss. Endothelial progenitor cell (EPC) deficiency and elevated asymmetrical dimethyl-L-arginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, predict adverse CAD outcome. Our aim was to assess changes in estimated glomerular filtration rate over time (DeltaeGFR) in relation to baseline EPC blood counts and ADMA levels in stable angina.Eighty non-diabetic men with stable angina were followed up for 2 years after elective coronary angioplasty. Exclusion criteria included heart failure, left ventricular systolic dysfunction, eGFR30 ml/min/1.73 m(2) and coexistent diseases. Those with cardiovascular events or ejection fraction55% during the follow-up were also excluded. A baseline blood count of CD34+/kinase-insert domain receptor (KDR)+ cells, a leukocyte subpopulation enriched for EPC, was quantified by flow cytometry (percentage of lymphocytes).A synergistic interaction (P = 0.015) between decreased CD34+/KDR+ cell counts and increased plasma ADMA, but not symmetrical dimethyl-L-arginine, was the sole significant multivariate DeltaeGFR predictor irrespective of baseline eGFR. DeltaeGFR was depressed in the simultaneous presence of high ADMA (0.45 micromol/l, median) and low CD34+/KDR+ cell counts (0.035%, median) compared to either of the other subgroups (P = 0.001-0.01). DeltaeGFR did not correlate with traditional risk factors, angiographic CAD extent, levels of C-reactive protein and soluble vascular cell adhesion molecule-1.Elevated ADMA and EPC deficiency may synergistically contribute to accelerated renal function decline in stable angina. This could result from the impairment of the EPC-dependent endothelial renewal in the kidney, an NO-dependent process.

Background Systemic lupus erythematosus (SLE) is characterized by increased cardiovascular morbidity and mortality. SLE patients have increased prevalence of subclinical atherosclerosis, although the mechanisms of this observation remain unclear. Considering the emerging role of monocytes in atherosclerosis, we aimed to investigate the relationship between subclinical atherosclerosis, endothelial dysfunction and the phenotype of peripheral blood monocytes in SLE patients. Methods We characterized the phenotype of monocyte subsets defined by the expression of CD14 and CD16 in 42 patients with SLE and 42 non-SLE controls. Using ultrasonography, intima-media thickness (IMT) of carotid arteries and brachial artery flow-mediated dilation (FMD) as well as nitroglycerin-induced dilation (NMD) were assessed. Results Patients with SLE had significantly, but only modestly, increased IMT when compared with non-SLE controls (median (25th/75th percentile) 0.65 (0.60/0.71) mm vs 0.60 (0.56/0.68) mm; p Conclusions Our observations suggest that CD14dimCD16+monocytes are associated with subclinical atherosclerosis in SLE, although the mechanism appears to be independent of endothelial dysfunction.

Human peripheral blood monocytes become apoptotic following phagocytosis and killing of Staphylococcus aureus . Although this type of monocyte apoptosis is known to be initiated by Fas-Fas ligand (FasL) interactions, the downstream signaling pathway has not been determined. In this work the involvement of mitochondria and the kinetics of caspase-8 and caspase-3 activation after phagocytosis of S. aureus were studied. Caspase-8 activity was measured in cell lysates by using the fluorogenic substrate Ac-IETD-AFC. Active caspase-3 levels and mitochondrial membrane potential (Δψ m ) were measured in whole cells by flow cytometry using monoclonal antibodies reacting with activated caspase-3 and chloromethyl-X-rosamine, respectively. The results show that caspase-8 was activated shortly after phagocytosis of bacteria. Caspase-8 activation was followed by progressive disruption of Δψ m , which is associated with the production of reactive oxygen intermediates. The irreversible caspase-8 inhibitor zIETD-FMK prevented the disruption of Δψ m and the release of cytochrome c from S. aureus -exposed monocytes. Caspase-3 activation occurred following disruption of Δψ m . These results strongly suggest that apoptosis of monocytes that have phagocytosed and killed S. aureus is driven by the Fas-FasL-initiated pathway, which is typical for type II cells.

The phenotype and function of peripheral blood monocytes change after trauma and during sepsis. The aim of the study was to evaluate monocyte expression of human leucocyte antigen (HLA)-DR and Fc receptor III (FcR III) (CD16) in neonates and small children with high risk of sepsis (hospitalized at the intensive care unit). The reduced proportion of CD14+HLA-DR+ monocytes was observed in all patients at the intensive care unit, while the increase of CD16 expression on monocytes was observed in the course of sepsis. The measurement of CD16 expression on monocytes also proved to be more useful for monitoring patient. The proportion of both CD14dimCD16+ and CD14highCD16+ monocytes increased during sepsis; however, monocytes showed reduced ability to phagocytose Escherichia coli, compromised ability to cooperate with T cells and reduced CD86 expression in parallel to HLA-DR depression. The reduced interleukin (IL)-1 but rather increased IL-10 production was associated with sepsis. The differences between CD14+CD16+ monocytes of healthy donors and patients with sepsis are discussed.

Phagocytosis and killing of microorganisms by reactive oxygen radicals are important defence mechanisms of the immune system and it was shown that human monocytes (MO) are heterogeneous in exerting these functions. Previously, we described that human peripheral blood MO consist of a major subset of Fcγ-receptor-I (CD64)-positive cells exhibiting low accessory cell capacity but high phagocytic activity, and a minor subset of CD64-negative cells with dendritic cell (DC)-like high T cell accessory cell capacity but low phagocytic capacity. Recently, we could show that each subset itself further differs in the expression of the Fcγ-receptor-III (CD16) and T cell accessory activities resulting in four different subsets: two CD16 + subsets (CD64 + or CD64 - ) with high T cell stimulation capacity and two CD16 - subsets (CD64 + or CD64 - ) with low accessory activities. In the present study we demonstrate that these subsets also differ in their ability to phagocytose opsonized bacteria ( S. aureus and E. coli ) and in the generation of reactive oxygen species. Both CD64 + subsets (CD16 + or CD16 - ) exhibit high phagocytic activity accompanied by intracellular superoxide induction. Luminal-dependent (mainly myeloperoxidase (MPO)-mediated) chemiluminescence (CL) response to latex and FMLP (formylmethionylleucylphenylalanine) was also high in these cell populations. Phagocytic activity and modest CL response was shown in CD64 - /CD16 + but not in CD64 - /CD16 - cells, indicating that each subset except for CD64 - /CD16 - cells may engulf bacteria and exhibit MPO activity. Taken together, these data demonstrate further heterogeneity of peripheral blood MO in both, phagocytic activity and generation of reactive oxygen species indicating differences between the four subsets in this kind of defence mechanisms against pathogens.

Human peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus . The consequences of heat stress for monocytes were studied with regard to the effect on S. aureus -induced apoptosis. Exposure of monocytes to 41.5°C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureus immediately or shortly after heat shock had no effect on the S. aureus -induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus -induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis.

During bacterial infections of the respiratory tract, neutrophils (PMN) are recruited to the lung by various mechanisms, including production of interleukin-8 (IL-8) by alveolar macrophages (AM). After fulfilling their defense function, PMN become apoptotic and have to be disposed of by AM to prevent local damage to the lung tissue by oxygen species and proteolytic enzymes. We measured the levels of IL-8 in the bronchoalveolar lavage (BAL) and the ability of AM to engulf senescent PMN in a groups of children with and without recurrent infections of the respiratory tract. The IL-8 level was measured by enzyme-linked immunosorbent assay (ELISA). The phagocytosis of apoptotic neutrophils was evaluated microscopically by the presence of myeloperoxidase positive material in AM before and after 1 h of incubation with senescent PMN. The data show that children suffering from recurrent infections have increased IL-8 in BAL and that their AM have a lower ability to engulf apoptotic PMN in vitro. Furthermore, the proportion of annexin V-binding cells was higher in BAL of children with recurrent infections of the respiratory tract than in normal controls. Pediatr Pulmonol. 1999; 27:167–173. © 1999 Wiley-Liss, Inc.

Efficient removal of apoptotic polymorphonuclear leukocytes (PMNs) is an important step in the resolution of inflammation, which protects tissues from the noxious contents of dying cells. While the impairment of apoptotic PMNs removal has been demonstrated for macrophages in systemic lupus erythematosus (SLE), recent studies show that monocytes are also capable of such phagocytosis, although their involvement in SLE is not clear. Therefore, we characterized phagocytosis of apoptotic PMNs by monocytes in 22 patients with SLE and 22 healthy controls. Using flow cytometry we demonstrate that in SLE peripheral blood monocytes show impaired phagocytosis of autologous apoptotic PMNs, while they efficiently engulf apoptotic PMNs isolated from healthy subjects. Monocytes CD14highCD16+ and CD14dimCD16+ more efficiently interacted with apoptotic neutrophils than CD16– cells both in SLE and healthy subjects. Monocytes in SLE showed modestly decreased expression of CD35 and CD91 and increased expression of T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3); however, these differences were evident mainly in selected subsets of monocytes (CD16+) while defects in phagocytosis were observed in all monocyte subsets. Apoptotic cell-dependent induction of lipopolysaccharide (LPS) stimulated production of anti-inflammatory cytokine IL-10 by peripheral blood mononuclear cells (PBMC) was blunted in SLE while the production of pro-inflammatory cytokine TNF-α was unchanged.

In this study the ability of soluble interleukin-6 receptor (sIL-6R) to stimulate interleukin-6 (IL-6) synthesis in human fibroblasts is described. It was found that sIL-6R, in combination with endogenous or exogenous IL-6, markedly upregulated IL-6 synthesis. These data suggest that increased IL-6 production after stimulation by either interleukin-1 or tumor necrosis factor-α would result in complex formation with sIL-6R, rapid uptake, and further synthesis of this cytokine. Furthermore, it would explain the decrease in sIL-6R plasma levels observed in patients suffering from sepsis.

We have previously shown that exogenous human recombinant tumour necrosis factor-alpha (rTNF), added before an antigen pulse, enhanced antigen presentation by human blood monocytes. The present study shows that, surprisingly, rTNF added after an antigen (PPD) pulse inhibited, while anti-TNF monoclonal antibody (mAb) enhanced, antigen presentation. mAbs htr-9 against p55 TNF receptor type I (TNF-RI) abrogated rTNF enhancing effect on PPD presentation and decreased presenting activity of untreated monocytes while utr-1 mAb, against p75 TNF receptor type II (TNF-RII), reversed the inhibitory effect of rTNF given after antigen pulse. PPD and rTNF when added singly induced TNF-mRNA accumulation in monocytes. Pretreatment of monocytes with rTNF followed by a PPD pulse caused and enhancement of TNF-mRNA accumulation. However, when post-treatment with rTNF was applied to PPD-pulsed monocytes, then inhibition of TNF gene expression was seen. This may point to the role of endogenously generated TNF in regulation of antigen-presenting capacity of monocytes. These studies indicate that TNF is an important regulator of monocyte antigen-presenting capacity and that the level of TNF gene activation in monocytes may be associated with their ability to present nominal antigen.

Human peripheral blood monocytes cocultured with tumour cells were used as an in vitro model of in situ interactions between tumour-infiltrating macrophages and the tumour. Tumour cells stimulated de novo expression of the human tumour necrosis factor alpha (TNF) gene in monocytes and caused the release of TNF into the culture supernatant. A group of 14 patients with stage IVA gastric cancer receiving adjuvant chemotherapy (5-FU, Adriamycin, mitomycin C: FAM) or immunochemotherapy (BCG+FAM) was investigated for the ability of monocytes to produce TNF in vitro upon stimulation with tumour cells or purified protein derivative of tuberculin (PPD). Patients were followed at biweekly intervals, i.e. before each instillation of BCG epicutaneously over a period of 10 weeks. It was found that monocytes of some patients receiving BCG at the end of the observation period had an enhanced ability to produce TNF following stimulation with tumour cells. In contrast, such production was not substantially altered during the study period in patients on chemotherapy. PPD-induced TNF production was much weaker and was not significantly changed during this observation time. We infer that BCG immunotherapy may induce the subtle changes in some cancer patients that lead to an increased interaction between monocytes and tumour cells and result in enhanced production of cytokine(s) with antitumour properties.

Aim:To assess endothelial progenitor cells (EPC) counts, a novel prognostic marker, in relation to classical adverse outcome predictors–N-terminal pro–B-type natriuretic peptide (NT-proBNP), impaired left ventricular (LV) relaxation and exercise-induced ischemia–in stable coronary artery disease (CAD) with preserved LV systolic function.Methods:We studied 30 non-diabetic men with one-vessel CAD, LV ejection fraction ≥ 60% and normal LV diastolic function (n= 16) or impaired LV relaxation (by ultrasound including tissue Doppler) (n= 14), and 14 non-CAD controls matched for risk profile and medication. CD34+/kinase-insert domain receptor (KDR)+ cells (CD34+/KDR+ cells), a leukocytes subpopulation enriched for EPC, were enumerated by flow cytometry.Results:CAD patients with abnormal LV relaxation exhibited significantly elevated NT-proBNP and decreased CD34+/KDR+ cells vs. CAD with regular diastolic function and non-CAD controls. An inverse NT-proBNP–CD34+/KDR+ cells relationship was precipitated by the clustering of high resting NT-proBNP and low CD34+/KDR+ cells in the subjects with a lower Duke treadmill score.Conclusions:Propensity to symptomatic exertional ischemia may underlie the coincidence of moderately elevated NT-proBNP and EPC deficiency in stable angina. Additionally, chronic subclinical ischemia can also be involved in these associations. These might result from BNP overexpression in the ischemic myocardium and a hypothetical exhaustion of the bone marrow capacity to mobilize EPC at multiple ischemic episodes, thus contributing to NT-proBNP prognostic effect irrespective of hemodynamic factors.

Macrophages have the potential to recognize apoptotic neutrophils and phagocytose them while the same function for monocytes is uncertain. In fact, early findings indicated that monocytes started to phagocytose neutrophils on the third day of differentiation to macrophages. Here we show, using flow cytometry and confocal microscopy, that peripheral blood monocytes phagocytose apoptotic but not freshly isolated granulocytes. Recognition of apoptotic cells is predominantly connected with CD16(+) monocytes (CD14(high) CD16(+) and CD14(dim) CD16(+)) and requires CD36. Clearance of apoptotic polymorphonuclear leucocytes appears to be independent of the CD14 mechanism. Uptake of apoptotic Jurkat T cells by monocytes is CD14 and CD36 dependent. Liposomes containing phosphatidyl-l-serine reduce binding of apoptotic polymorphonuclear leucocytes. Lipopolysaccharide-activated subpopulations of monocytes while in contact with apoptotic cells produce more anti-inflammatory cytokine interleukin-10 whereas the production of pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta is reduced.

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.

Objectives: Angiogenesis, the process of formation of blood vessels, is essential for many physiological as well as pathological processes. It has been shown that human adipose tissue contains a population of non‐characterized cells, called stromal‐vascular fraction (SVF) cells, which are able to differentiate into several lineages. The aim of this study was to determine conditions for promoting differentiation of human adipose tissue progenitors towards endothelial cells, as well as to show that SVF cells cooperate with differentiated endothelium in capillary network formation. Materials and methods: Stromal vascular fraction cells were isolated according to modified Hauner's method and after adaptation they were cultured in pro‐angiogenic or pro‐adipogenic medium. Cells were characterized by presence of surface antigens by flow cytometry, and by expression of genes characteristic for endothelial cells or for adipocytes, quantitative real‐time polymerase chain reaction. A number of tests were performed to verify their differentiation. Results: Differentiation of human SVF cells towards endothelium was stimulated by the presence of serum and absence of adipogenic factors, documented by the pattern of gene expression as well as different functional in vitro assays. SVF cells were found to work together with human umbilical vein endothelial cells to form capillary networks. Conclusions: Here, we show that differentiation of SVF cells to endothelial cells or adipocyte‐like cells depended on the medium used. Our work provides a clear model for analysing the differentiation capacity of SVF cells.

BACKGROUND Low blood counts of CD34/kinase-insert domain receptor double-positive cells (CD34(+)/KDR(+) cells)-a leukocytes subpopulation enriched for bone marrow-derived endothelial progenitor cells (EPC)- predict adverse outcomes in coronary artery disease (CAD). The dependence of EPC numbers on the glomerular filtration rate (GFR), another prognostic factor, has not been reported in CAD yet. Our aim was to assess CD34(+)/KDR(+) cell counts versus GFR in stable angina. METHODS We studied 102 stable angina men with severe angiographic CAD and normal left-ventricular systolic function. CD34(+)/KDR(+) cells were enumerated by flow cytometry. RESULTS With lowering GFR, CD34(+)/KDR(+) cell numbers (% of lymphocytes, median and interquartile range) decreased: 0.04 (0.03-0.06), 0.03 (0.02-0.05) and 0.02 (0.01-0.03)% for GFR >or=90, 60-89 and 30-59 ml/min/1.73 m(2), respectively (P < 0.001 for trend). CD34(+)/KDR(+) cell counts correlated with GFR (r = 0.25, P = 0.01), CAD extension score (r = -0.20, P = 0.04), soluble form of vascular cell adhesion molecule-1 (sVCAM-1) (r = -0.22, P = 0.03) and homocysteine (r = -0.20, P = 0.04) levels. A GFR

Based on CD14 and CD16 expression, human peripheral blood monocytes (MO) can be divided into a major CD14(high) CD16(-) population and two minor CD14(high) CD16(+) and CD14(dim) CD16(+) subpopulations. CD14(dim) CD16(+) MO are well characterized and regarded as pro-inflammatory because upon stimulation produce TNF-alpha but little, if any, IL-10. By contrast, little is known about CD14(high) CD16(+) MO. We investigated the surface expression of selected determinants by CD16(+) MO subpopulations, cytokine production, phagocytosis and antigen presentation. We found that both CD16(+) subpopulations had a higher expression of HLA-DR, CD86, CD54 and a lower expression of CD64 than CD14(high) CD16(-) population. In addition, CD14(high) CD16(+) MO showed a higher expression of CD11b and TLR4 than CD14(dim) CD16(+) and CD14(high) CD16(-) subpopulations. CD14(high) CD16(+) MO exhibited an increased phagocytic activity and a decreased antigen presentation in comparison with CD14(dim) CD16(+). As expected, lipopolysaccharide (LPS)-stimulated CD14(dim) CD16(+) MO produced TNF-alpha but little IL-10. By contrast, LPS-stimulated CD14(high) CD16(+) subpopulation produced significantly more IL-10 than CD14(dim) CD16(+) and CD14(high) CD16(-) MO. In conclusion, our data show that human peripheral blood CD16(+) MO are heterogeneous in function and consist of two subpopulations: CD14(dim) CD16(+) pro-inflammatory and CD14(high) CD16(+) with anti-inflammatory potential.

While immunopathogenesis of atherosclerosis is well defined, immunological mechanisms of hypertension (HTN) and its link to atherosclerosis remain unclear. We hypothesized, that primary HTN is associated with a pro-inflammatory and pro-atherogenic phenotype of circulating monocytes, characterized by co-expression of the Fc-gamma receptor (CD16+) and LPS receptor (CD14+high). Methods: 132 subjects (74M and 58F) with typical clinical risk factors of atherosclerosis were studied. HTN (85%) was diagnosed based on blood pressure monitoring and use of anti-HTN drugs. Monocyte characteristics in the peripheral blood were determined by flow cytometry and plasma cytokine levels by ELISA. Results: CD16+CD14+ cells were more prevalent in subjects with HTN and were highest in subjects whose blood pressure was poorly controlled (Figure ). A significant correlation between blood pressure levels at the time of study (JNC7) and prevalence of CD14+CD16+ monocytes was found (Figure ). These relationships remained significant in multi-variate analysis (p = 0.01; type 3 sum of squares ANOVA) taking into account other major risk factors for atherosclerosis or treatments. Further analysis of subpopulations showed that levels of CD16 +CD14+ (high) monocytes, rather than CD16 + CD14(dim) revealed a strong relationship to hypertension. These monocytes showed significantly higher expression of activation markers HLA-DR and CD45RA and TNF-α production(p

Objective Similarities between rheumatoid arthritis (RA) and atherosclerosis include endothelial dysfunction (an antecedent of plaque formation) and depletion of circulating bone marrow–derived endothelial progenitor cells. This study was undertaken to test the hypothesis that endothelial progenitor cell depletion and subclinical atherosclerosis in RA may be related to accumulation of an endogenous inhibitor of nitric oxide (NO) synthesis, asymmetric dimethyl-L-arginine. Methods We studied 30 patients with active RA and 20 age- and sex-matched healthy controls. Exclusion criteria were clinically evident atherosclerosis, traditional risk factors, hyperhomocysteinemia, and renal dysfunction. The blood endothelial progenitor cell count was assayed by flow cytometry and expressed as a percentage of lymphocytes. Plasma L-arginine, asymmetric dimethyl-L-arginine, and symmetric dimethyl-L-arginine were measured with liquid chromatography–mass spectrometry. Mean carotid intima-media thickness (IMT) was assessed by B-mode ultrasound. Results In RA patients, we found elevated levels of asymmetric dimethyl-L-arginine (mean ± SD 0.49 ± 0.07 μmoles/liter versus 0.40 ± 0.07 μmoles/liter in controls; P < 0.001), a depressed endothelial progenitor cell count (0.039 ± 0.025% versus 0.063 ± 0.035%; P < 0.05), and increased IMT (0.65 ± 0.13 mm versus 0.55 ± 0.10 mm; P < 0.01), with no differences in levels of L-arginine or symmetric dimethyl-L-arginine. The endothelial progenitor cell count was inversely correlated with the level of asymmetric dimethyl-L-arginine. IMT was positively related to the ratio of asymmetric dimethyl-L-arginine to L-arginine and negatively related to the endothelial progenitor cell count, in univariate and multivariate analyses. Conclusion Plasma asymmetric dimethyl-L-arginine levels are elevated in RA patients free of cardiovascular disease or risk factors. Asymmetric dimethyl-L-arginine accumulation may contribute to endothelial progenitor cell depletion via depressed NO-dependent endothelial progenitor cell mobilization and/or survival, with consequent impairment of endothelial progenitor cell–mediated endothelial repair, which can promote atherogenesis in RA.

Background: Internucleosomal DNA fragmentation is one of the hallmarks of apoptosis. Because the low molecular weight DNA fragments are extracted during cell staining in aqueous solutions, apoptotic cells can be identified on DNA content frequency histograms as cells with fractional (“sub-G1”) DNA content. The aim of the present study was to explore whether in situ DNA fragmentation during apoptosis is discontinuous or progresses incessantly and if it is discontinuous, to define the resistant to cleavage fraction of DNA that remains stainable with the fluorochrome. Materials and Methods: The model of activation-induced apoptosis of human lymphocytes was chosen as it provides uniform cell population with identical DNA content (DI = 1.00) that undergo apoptosis. Their apoptosis was induced by multivalent mitogen phytohemagglutinin (PHA) in the absence and presence of geldanamycin (GA), the benzoquinone ansamycin antibiotic which binds to Hsp90 (Heat Shock Protein 90) and alters its function. The cells were stained with acridine orange, the metachromatic fluorochrome that differentially stains cellular DNA and RNA. Results: A sharp, discrete peak representing the subpopulation of “sub-G1” cells with highly reproducible DI = 0.42 ± 0.02 (CV = 5.5 ± 1.2) was observed on DNA content histograms of lymphocytes whose apoptosis was induced by PHA alone. Two distinct peaks, one representing cell subpopulations with DI = 0.42 (as above) and another, with DI = 0.79 ± 0.04 (CV = 5.8 ± 0.4), respectively, were seen in apoptotic cells from cultures stimulated with PHA in the presence of GA. The frequency of cells represented by the sub-G1 peaks varied depending on time of induction of apoptosis and GA concentration. Conclusions: Apoptosis-induced DNA fragmentation is discontinuous; approximately 42% of DNA is relatively stable and remains within the cell. The data suggest that the stable DNA is associated with nuclear matrix while the degradable fraction represents DNA in loop domains. A transient DNA stabilization is apparent in the presence of GA as evidenced by the presence of cell subpopulations with 79% of DNA retained in the cell. The observed discontinuity of DNA fragmentation appears to reflect sequential involvement of different nucleases and may also be modulated by chromatin structure. © 2007 International Society for Analytical Cytology.

To determine whether monocytes can be generated from CD34+ hematopoietic progenitors in large numbers, cord blood CD34+ cells were first expanded for 3–10 days in X-VIVO 10 medium supplemented with FCS, stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 Ligand (Flt-3L), and then differentiated in IMDM medium supplemented with FCS, SCF, Flt-3L, IL-3 and M-CSF for 7–14 days. These two step cultures resulted in up to a 600-fold mean increase of total CD14+ cells. Using this approach, two subpopulations of monocytes were obtained: CD14+CD16− and CD14++CD16+ occurring at 2:1 ratio. 1.25(OH)2 Vitamin D3 added to the differentiation medium altered this ratio by decreasing proportion of CD14++CD16+ monocytes. In comparison to CD14+CD16−, the CD14++CD16+ cells showed different morphology and an enhanced expression of CD11b, CD33, CD40, CD64, CD86, CD163, HLA-DR, and CCR5. Both subpopulations secreted TNF and IL-12p40 but little or no IL-10. CD14++CD16+ monocytes released significantly more IL-12p40, were better stimulators of MLR but showed less S. aureus phagocytosis. These subpopulations are clearly different from those present in the blood and may be novel monocyte subsets that represent different stages in monocyte differentiation with distinct biological function.

Every human being is genetically unique and this individuality is not only marked by morphologic and physical characteristics but also by an individual's response to a particular drug. Single nucleotide polymorphisms (SNPs) are largely responsible for one's individuality. A drug may be ineffective in one patient, whereas the exact same drug may cure another patient. Recent advances in DNA mapping and other screening technologies have provided researchers and drug developers with crucial information needed to create drugs that are specific for a given individual. In the future, physicians will be able to prescribe individualised drugs adjusted to, for example, activities of specific enzymatic pathways that would either be targeted by these drugs, or would be responsible for drug conversion or inactivation. Furthermore, the mapping of the human genome allows broader development and application of drugs that act on the level of gene transcription rather than as simple biochemical inhibitors or activators of certain enzymes. Such new approaches will maximise desired therapeutic results and may completely eliminate severe side effects. To illustrate the potential of genetic translational research, the authors discuss available analytical methodologies such as; gene arrays, flow cytometry-based screening for SNPs, proteomics, metabolomics, real-time PCR, and other methods capable of detecting both SNPs, as well as more profound changes in cell metabolism. Finally, the authors provide several examples that focus mostly on targeting protein-DNA interactions, but also other processes.

Angiogenesis is a crucial process in tissue remodeling during growth, both in the embryo and the adult. In our study we concentrated on the direct effect of β-carotene on human umbilical cord originating from endothelial progenitor cells (EPCs). β-Carotene uptake by EPCs was measured using a HPLC method. The determination of cell surface antigens was performed by flow cytometry. The effect on cell proliferation was estimated by measuring bromo-deoxyuridine incorporation. The influence on the formation of a tubular-like structure was investigated in a 3D assay in matrigel. Quantitative gene expression was estimated using real-time PCR. We demonstrated that β-carotene in the physiological range of concentrations found in human blood is a potent activator of EPC chemotaxis, which is accompanied by a change in the expression of genes mediating cell adhesion and homing, but does not activate the final markers of endothelial differentiation. This study points to the prochemotactic and homing activity of β-carotene in undifferentiated endothelial cell progenitors for the first time, which may suggest a potential role of this carotenoid in progenitor cell therapy aimed at angiogenesis and tissue repair.

Summary Background Staphylococcus aureus has important implications for the pathogenesis of atopic dermatitis (AD). In some patients S. aureus can be eradicated from the skin during anti-inflammatory treatment, while in others bacterial colonization is persistent. Potential mechanisms and features of these two distinct groups of patients are not known. Objective Accordingly, we studied relationships between the ability to eliminate S. aureus during an anti-inflammatory treatment and selected clinical and immunological features. Methods Quantitative assessment of S. aureus on the skin, in nasal vestibule and throat, serum IgE levels, CD4/CD8 T-cell ratio, lymphocyte proliferation and phagocyte oxidative burst were determined during the exacerbation and after 4 and 12 weeks of the treatment using topical steroid and oral antihistamine in 34 patients with AD. Results S. aureus was found on the skin of all 34 patients during exacerbation. Disease severity scoring of atopic dermatitis (SCORAD) correlated with the density of bacteria. Treatment with oral antihistamine and topical steroid resulted in a significant alleviation of symptoms, which correlated with the elimination of S. aureus from the skin in 70% of patients. In the remaining 30% of patients, dense (more than 1010/cm2) S. aureus skin colonization, persisted despite the treatment. Patients with persistent S. aureus presented with higher serum IgE levels, lower lymphocyte proliferation in response to staphylococcal enterotoxin B, phytohaemagluttinin and anti-CD3. Persistence of S. aureus was more common in men. Conclusions Patients with AD differ in the ability to clear S. aureus from the skin during anti-inflammatory treatment, which appears to be related to the abnormalities in immunological parameters. Local antibiotic therapy should be considered only in patients with persistent S. aureus colonization.

Despite significant progress in cancer therapy, the outcome of the treatment is often unfavorable. Better treatment monitoring would not only allow an individual more effective, patient-adjusted therapy, but also it would eliminate some of the side effects. Using a cytochrome c ELISA that was modified to increase sensitivity, we demonstrate that serum cytochrome c is a sensitive apoptotic marker in vivo reflecting therapy-induced cell death burden. Furthermore, increased serum cytochrome c level is a negative prognostic marker. Cancer patients whose serum cytochrome c level was normal 3 years ago have a twice as high probability to be still alive, as judged from sera samples collected for years, analyzed recently and matched with survival data. Moreover, we show that serum cytochrome c and serum LDH-activity reflect different stages and different forms of cell death. Cellular cytochrome c release is specific for apoptosis, whereas increased LDH activity is an indicator of (secondary) necrosis. Whereas serum LDH activity reflects the "global" degree of cell death over a period of time, the sensitive cytochrome c-based method allows confirmation of the individual cancer therapy-induced and spontaneous cell death events. The combination of cytochrome c with tissue-specific markers may provide the foundation for precise monitoring of apoptosis in vivo, by "lab-on-the-chip" technology. (c) 2005 Wiley-Liss, Inc.

We asked whether in atopic dermatitis (AD) increased T cell apoptosis in staphylococcal enterotoxin B (SEB)-activated cultures of peripheral blood mononuclear cells (PBMCs) is characteristic of the exacerbation of the disease or connected with skin colonization by Staphylococcus aureus.The clinical status of the patients was evaluated using the SCORAD index. The number of bacteria colonizing patients' skin lesions was determined by the cfu method. Mononuclear cells isolated from peripheral blood were stimulated by SEB and the apoptosis of CD3+ cells in culture was determined by flow cytometry using the monoclonal antibody APO2.7. The cytokine production in the culture supernatants was determined by ELISA and Cytometric Bead Array kits.T cell apoptosis was increased, while the production of interferon (IFN)-gamma was reduced in cultures of PBMCs of AD patients during exacerbation. The proportion of CD3+ APO2.7+ cells positively correlated with the density of S. aureus recovered from skin lesions, but not with SCORAD index. By contrast, SCORAD index, but not S. aureus density, negatively correlated with IFN- gamma production. Furthermore it was found that the presence of S. aureus on uninvolved skin distinguishes a group of severe cases with high serum IgE level, increased T cell apoptosis, and reduced production of tumor necrosis factor alpha in SEB- -stimulated cultures.Among AD patients the increased activation-induced T cell apoptosis observed in SEB- -stimulated cultures is related to skin colonization by S. aureus. The presence of bacteria on uninvolved skin is a feature of a distinct group of AD patients.

During acute inflammation, leukocyte infiltration is mostly neutrophilic, but later monocytes prevail. The majority of inflammatory cells, particularly neutrophilic polymorphonuclear leukocytes (PMNs), become apoptotic at later stages of inflammation and are phagocytosed by neighboring cells, mostly by macrophages. Recently, it has been found that human peripheral blood monocytes also recognize apoptotic cells, which primes them to increased production of interleukin (IL)-10--a cytokine known to reduce phagocytes' ability to engulf and kill pathogens. Based on the above, we studied monocytes' ability to phagocytose and kill Staphylococcus aureus while in contact with apoptotic cells.Monocytes isolated by elutriation were co-cultured with apoptotic PMNs or Jurkat cells and exposed to viable, human serum-opsonized S. aureus. To induce apoptosis PMNs were cultured overnight while Jurkat cells were UV-treated. Apoptosis, phagocytosis of bacteria and intracellular superoxide production were measured by flow cytometry. Production of reactive oxygen species was also followed by measurement of chemiluminescence. The bactericidal effect was determined by standard colony forming units method.Data presented show that contact of monocytes with apoptotic neutrophils and Jurkat cells had no influence on monocyte phagocytosis of S. aureus, the generation of reactive oxygen species, or the killing of bacteria.The data obtained suggest that monocytes attracted to the inflammatory site are not deficient in their ability to cope with pathogens after contact with apoptotic cells despite increased production of IL-10.

Pathogenesis of atopic dermatitis (AD) is multifactorial, therefore despite many different treatment protocols none of the available methods is effective enough. In the present study we analysed the effects of oral antihistamines and topical steroids on selected parameters of humoral (immunoglobulin levels) and cellular (lymphocyte proliferation index, granulocyte oxidative burst, lymphocyte phenotype CD4:CD8) immunity. These parameters were analysed in 34 AD subjects during the exacerbation of the disease and after 4 and 12 weeks of the treatment.Along with the clinical status improvement (change form 3.17 +/- 0.1 to 1.3 +/- 0.2; 0-4 scale; p0.001), total serum IgE levels decreased significantly from 1563 +/- 459 to 1266 +/- +/- 364 IU/ml (p0.05). Other immunoglobulin levels did not change. Total blood chemiluminescence in response to latex stimulation was relatively higher during exacerbation than during remission, but this difference was not significant (p = 0.1). Mean spontaneous or PMA stimulated chemiluminescence did not differ either. CD4:CD8 index decreased significantly during the treatment (from 2.7 +/- 0.3 to 1.8 +/- 0.2; p = 0.03). Lymphocyte proliferation in response to PHA was significantly lower during exacerbation than during remission (10.4 +/- 2.8 vs. 27.1 +/- +/- 5.2; p0.03) and the improvement was observed after first 4 weeks of the treatment reaching the levels found in healthy controls. Proliferation index in response to anti-CD3 mAb (OKT-3) was in turn significantly higher during exacerbation (19.1 +/- 3.4 vs. 11.2 +/- 2.0; p = 0.04).Oral antihistamine in combination with topical steroids leads to several significant changes in the immune parameters (IgE levels, CD4:CD8 and proliferation indexes) which may explain its high effectiveness in majority of patients with AD.

Monocytes/macrophages undergo apoptosis and are in contact with apoptotic cells both in vitro and in vivo. The data show that monocytes undergoing spontaneous apoptosis in vitro change their cytokine production profile. We demonstrate that the lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10) is up-regulated, while production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is either not affected or reduced. These differences seen both at the protein and mRNA level directly correlate with the appearance of apoptotic cells in the culture. Flow cytometry analysis using double staining, surface with annexin V and intracellular with anti-IL-10, suggested that annexin V-negative monocytes are the predominant source of IL-10. Analysis of sorted populations of monocytes indicated that the increase in IL-10 synthesis appears to result from direct interactions between non-apoptotic and apoptotic cells at the time of stimulation. Also non-apoptotic, freshly isolated monocytes produced more IL-10 upon stimulation with LPS, Staphylococcus aureus or zymosan when apoptotic neutrophils were added to the culture. In contrast, monocyte-derived macrophages did not produce more IL-10 in the presence of apoptotic neutrophils. Finally, we found that the presence of apoptotic monocytes in the culture may influence specific immune responses. The data show that in the presence of annexin V-positive monocytes CD4-positive memory T cells produce less IFN-gamma upon stimulation with purified protein derivative of tuberculin, which could be partially reversed by anti-IL-10 neutralizing antibodies. We conclude that these findings might illustrate the mechanisms operating within an inflammatory site and play an important immunoregulatory role during the resolution of inflammation and specific immune responses.

Activated platelets adhere to the endothelium and release vasoactive mediators which induce vasoconstriction and remodeling of the vessel wall. The influence of native and ex vivo oxidized lipoproteins enriched with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC), the major lipid responsible for the biological activity of minimally oxidized LDL (mm-LDL), on platelet adhesion, membrane receptor expression and aggregation was studied. Influence of native and oxidized lipoproteins (5-100 microg protein/ml); ox-PAPC (0.5-50 microg/ml); ADP (1-10 microM) as well as the specific phosphatase 1 and 2A inhibitor okadaic acid (3-10 microM) on platelet adhesion, receptor expression and aggregation was measured. Platelets adhered to all the classes of lipoproteins immobilized in plastic microtiter wells (native lipoproteins: HDLLDLVLDLoxidized lipoproteinsox-PAPC-enriched lipoproteins). Flow cytometry revealed that lipoproteins increased CD41 expression. Preincubation of platelets with ox-PAPC alone, significantly up-regulated CD62p and CD41 receptors (higher dose) but potently inhibited anti-CD36 MoAb binding. Okadaic acid increased anti-CD41 and decreased anti-CD36 and anti-CD42b MoAbs binding. Neither ox-PAPC nor okadaic acid induced platelet aggregation. CD36 seems to be the main receptor responsible for binding of oxidized lipoproteins, particularly its ox-PAPC epitope. The effect of okadaic acid on CD36 and CD41 argue for the participation of phosphorylation-dependent reorganization of cellular trafficking and microtubule organization by ox-PAPC.

Human peripheral blood monocytes become apoptotic following phagocytosis ofStaphylococcus aureus. In this study, we investigated the mechanisms involved in this phenomenon. Cells exposed to bacteria were examined for the surface expression of Fas and Fas ligand (FasL). The level of soluble form of FasL was also measured in the culture supernatants. As Fas-mediated apoptosis involves the activation of caspases, the activities of caspase-8 and caspase-3 were determined. Finally, the involvement of oxidative stress in apoptosis of infected monocytes was investigated. The data indicated that as a consequence of phagocytosis ofS. aureus, FasL is released from the monocyte surface and induces apoptosis of phagocytic monocytes and to some extent the bystander cells. The importance of this mechanism was confirmed by demonstrating that blockage of CD95 preventsS. aureus-induced apoptosis of monocytes. Cell death occurring after phagocytosis ofS. aureusinvolves the activation of caspase-3-like proteases, as the specific caspase-3 inhibitor suppressed apoptosis of infected cells. The generation of reactive oxygen intermediates by phagocytic monocytes by itself is not sufficient as a death signal but rather acts in up-regulating FasL shedding and possibly in modulating caspase activity.

It has been demonstrated that in monocyte/T cell co-cultures activated with recall antigens, cytotoxic T cells were generated which are able to reduce the number of antigen-presenting monocytes. In previous studies we could show that a minor subset of monocytes, the Fc gamma receptor I-negative (CD64(-)) monocytes, exhibits significantly higher antigen-presenting capacity than the main population of monocytes (> 90%) which are Fc gamma receptor I-positive (CD64(+)). Therefore, we addressed the question whether they are also differentially susceptible to T cell-mediated killing. In the present study we demonstrate that the CD64(-) monocyte subset is more resistant to killing by antigen-activated T cells than CD64(+) monocytes, as indicated by a higher viability and recovery of CD64(-) monocytes. This mechanism involves CD95 (Fas) antigen, since monocyte death in co-cultures with antigen-activated T cells could be partially reduced by blocking anti-Fas monoclonal antibodies (mAb). In agreement with this finding, although CD95 antigen was expressed on CD64(+) and CD64(-) monocytes at comparable levels, killing of CD64(-) monocytes by activating anti-Fas mAb was lower than of CD64(+) monocytes.

The HIV-1 gp120 recombinant protein fragment encompassing aa residues 410-511, that contains the CD4 binding region (rp120cd), and fragment aa 446-511, which lacks the sequence responsible for CD4 binding (rp120), were synthesized to study their ability to induce TNF synthesis in human monocytes. The rp120cd stimulated TNFα secretion by monocytes while the rp120 and full-length recombinant protein (FL gp120), used as control, failed to do so. However, FL gp120 stimulated peripheral blood mononuclear cells (PBMC) and lymphocytes for TNF production and this was inhibited by anti-CD4 MAb. The rp120cd also caused TNF secretion by PBMC that was not blocked by this antibody. Furthermore, FL gp120 but not rp120cd inhibited anti-CD4 mAb binding to CEM cells. Hence, FL gp120 may cause TNF release from lymphocytes by binding to CD4, while rp120cd interacts with monocytes but not lymphocytes and induces TNF production by a mechanism not involving CD4 binding. Unexpectedly, FL gp120 but not rp120cd stimulated IL-6 secretion and IL-6 mRNA synthesis in monocytes. The FL gp120-induced production of IL-6 by monocytes was inhibited by anti-CD4 monoclonal antibody (MAb). Thus, there may be different requirements for TNF induction in lymphocytes and monocytes stimulated with various preparations of gp120 and for the selective induction of cytokines in monocytes. The enhanced production of TNF in HIV infection and AIDS may involve distinct cellular sources and different mechanisms.

In this study we investigated the effect of an in vitro infection with Mycobacterium tuberculosis on the ability of human monocytes to present the soluble antigen tetanus toxoid to T cells. We observed that tetanus toxoid-specific T-cell proliferation was markedly reduced when monocytes were infected with large numbers (bacterium-to-monocyte ratio, 50:1) of both viable and heat-killed mycobacteria. The level of antigen-induced gamma interferon release also was decreased when M. tuberculosis-containing monocytes were used as antigen-presenting cells. However, mycobacterium-infected monocytes did not show or trigger suppressive activity, because the presence of mycobacterium-infected monocytes did not affect the T-cell response induced by tetanus toxoid-pulsed control monocytes. When M. tuberculosis-infected monocytes were fixed with paraformaldehyde, they were not able to serve as antigen-presenting cells even in the presence of untreated accessory monocytes. Moreover, the uptake of both viable and heat-killed M. tuberculosis cells reduced the expression of human leukocyte antigen DR on monocytes. With regard to accessory function, monocytes infected with large numbers of mycobacteria were less efficient as accessory cells than were control monocytes in cultures of T cells activated with pokeweed mitogen. These results indicate that infection with large numbers of M. tuberculosis cells impairs the ability of monocytes to process and/or present soluble antigen and to serve as accessory cells in T-cell activation.

Nitric oxide (NO) has been recently identified as a potent mediator of tumoricidal activity of activated macrophages. Macrophages can be activated for tumor cell killing by microbial products, including lipopolysaccharide (LPS) and various cytokines. Here we report that in contrast to mouse macrophages, human peripheral blood monocytes stimulated with cytokines or LPS failed to release NO. Also priming of monocytes with interferon-gamma followed by activation with cytokines or LPS did not cause NO secretion. However, monocytes responded with NO production to stimulation with some human cancer cells but not with untransformed cells. NO production by monocytes was inhibited by NG-monomethyl-L-arginine, specific inhibitor of NO synthase and emetine, an irreversible blocker of protein synthesis. This may imply that human monocytes are unique in their restricted capacity to produce NO following interaction with some tumor cells, but not with other stimulators, and in this respect they may be able to distinguish between malignant and normal cells.

Pyocyanine, a pigment produced by Pseudomonas aeruginosa, has dual dose-dependent stimulatory as well as inhibitory effects on immune responses in vitro as measured by DNA synthesis of human T and B lymphocytes, interleukin-2 (IL-2) production by human T lymphocytes, immunoglobulin production by human B lymphocytes, and monokine production by human monocytes. In general, stimulatory activity was found at low concentrations of pyocyanine, whereas high concentrations of the pigment resulted in an inhibition of responses. At a pyocyanine concentration of 0.1 micrograms/ml or less the proliferation of T and B lymphocytes was enhanced, but at 0.5 micrograms/ml it was suppressed. IL-2 production by T lymphocytes was enhanced at concentrations up to 0.5 micrograms/ml but totally inhibited at 1.0 micrograms/ml. The differentiation of B lymphocytes to become immunoglobulin-producing cells was also enhanced in the presence of low doses of pyocyanine, whereas secretion of immunoglobulin by B lymphocytes was suppressed at all concentrations of pyocyanine. In contrast to the dual effects of pyocyanine on lymphocyte response, lipopolysaccharide-induced IL-1 and tumor necrosis factor release by monocytes was markedly enhanced by low as well as high concentrations of pyocyanine. From these results we conclude that this property of pyocyanine may lead to suppression of specific defense mechanisms and enhance harmful inflammatory reactions of the host during infection with Pseudomonas aeruginosa.

Imbalances in T cell subpopulations have been reported in multiple sclerosis (MS). In the present study of 31 MS patients, the percentage of T cells with Fc receptors for IgG (Tg) was found to be increased in patients with chronic progressive disease, and another T cell subset binding to the Raji B lymphoid cell line was decreased. An inverse correlation (r = -0.675; < 95% confidence limit) was found between these two subsets, suggesting that they vary inversely in MS. The mitogenic responses of MS mononuclear cells, isolated T cells, and recombinet T and non-T cells to the lectins phytohemagglutinin and pokeweek mitogen (PWM) did not differ from those of normal cells. However, more immunoglobulin (Ig)-producing cells were generated in a PWM-driven system with cells from MS patients than with cells from age-matched controls (p < 0.05). Autologous recombination of separated T and non-T cells did not significantly modify these results. T cells from MS patients added to B cells from normal controls exerted an effect that was related to their percentage of Tg cells; that is, values above 15% were associated with a suppression of Ig production, whereas for Tg values below 12%, a helper effect or no modification was observed. These results suggest that changes in T cell subsets in MS are related to changes in functional ability to modulate Ig production by normal B cells. However, MS B cells partly escape regulation by their own T cells, suggesting an associated B cell hyperactivity.

FcR+ and FcR- monocyte subsets were added to the pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I-stimulated cultures of peripheral blood mononuclear cells (PBMC) or to PBMC depleted of monocytes. The numbers of immunoglobulin-secreting cells (ISC) and cells with intracytoplasmic immunoglobulins (PC) were evaluated 6 days later. The addition of FcR- subset increased the number of ISC in cultures of PBMC stimulated with PWM and reconstituted the response of monocyte depleted PBMC. In contrast, FcR+ monocytes suppressed PWM-induced response and, when added in high dose, also that induced by S. aureus. The FcR+ monocytes suppressed the response by inhibition of immunoglobulin secretion but not the development of PC. This suggests that FcR+ monocytes may modulate humoral response by preferential inhibition of the final differentiation of B lymphocytes into ISC.

Methisazone, given subcutaneously to mice of BALB(c)W and Swiss strains in protective doses against lethal infection of vaccinia virus, showed immunosuppressive effects on humoral and cellular respons

Peripheral blood monocyte (MO) subpopulations isolated on a basis of functional expression or a lack of Fc receptor (FcR+ and FcR− MO) were used to study the regulation of the antigen (PPD)-driven lymphoproliferation. Long-term cultures of T lymphocytes with FcR+ MO, but not FcR− MO, led to the induction of T suppressor (Ts) cells that inhibited antigen-driven lymphoproliferation in the test cultures. These Ts cells were resistant to mitomycin C, belonged to afferent-acting category of Ts cells, expressed CD8 and HLA-DR determinants, and showed no antigenic specificity nor genetic restriction in action. The expression of MHC class II molecules (HLA-DR and HLA-DP but not HLA-DQ determinants) on MO which were used for antigen presentation was critical for Ts cell induction. It was concluded that specialized MO subpopulations may regulate the lymphoproliferation by inducing Ts cells (or Ts cell circuit) that in turn inhibit antigen-driven immune response.

Streptolysin S, which was found to be cytotoxic for mouse and human lymphocytes and particularly for their T subpopulation, was also shown to affect some of the functions ascribed to T lymphocytes. In vitro studies demonstrated that streptolysin S-pretreated lymphocytes exhibited reduced responsiveness to phytohemagglutinin and decreased lymphokine production. Streptolysin S could also alter the immune response of mice in vivo. It induced suppression of the immune response to T-dependent antigen (SRBC) but had not influence on response to T-independent antigen (S III). The in vitro and in vivo studies suggest that streptolysin S can impair the function of T lymphocytes or, more precisely, of some subpopulation of T cells.

SOME highly polymerised immunogens with repeated antigenic determinants, such as bacterial polysaccharides or polymerised flagellin and hapten conjugates of these, can stimulate mouse B lymphocytes to make antibody without requiring the cooperative helper activity of T lymphocytes1. The mechanism whereby such T-independent immunogens, after interaction with the surface receptors on specifically responsive B lymphocytes, trigger these into activity is uncertain. Dukor and Hartmann2 have postulated that binding of activated C3 to complement receptors on B cells acts as a necessary second signal for antibody production. They envisage that T-independent antigens and B-cell mitogens generate this second signal by their ability to activate C3 through the ‘bypass’ pathway, whereas in the case of T-dependent antigens the signal derives from C3 cleavage by proteases released by activated T cells.

The spontaneous migration in vitro, production of T cell migration inhibitory factor (TIF) and the response to TIF of OKT4+ and OKT8+ human T cell subsets were studied. The OKT4+ lymphocytes migrated far better than the OKT8+ cells although the movement of both subsets was comparably inhibited by TIF. The OKT8+ subset was found to be a major source of TIF, while OKT4+ cells were responsible for macrophage migration inhibitory factor (MIF) production. The implications of lymphokine production by OKT8+ cells for the regulation of inflammatory responses are discussed.

In coculture experiments with normal lymphocytes, peripheral blood lymphocytes (PBL) of 10 boys with hypogammaglobulinemia were screened for the presence of cells able to suppress Pokeweed mitogen (PWM)- and Staphylococcus aureus Cowan I-induced immunoglobulin production in vitro . PBL and T lymphocytes of two patients were shown to suppress reproducibly PWM-induced immunoglobulin production of control PBL and of control B + T lymphocytes. PBL of three other patients were also able to suppress but their activity was expressed only in combination with some but not other normal lymphocytes. In neither case was the Cowan I-induced response suppressed. PBL and T lymphocytes of one other patient were able to suppress both PWM- and S. aureus Cowan I-induced immunoglobulin production of normal lymphocytes. These data provide evidence for two functionally distinct suppressor T lymphocytes in hypogrammaglobulinemic patients.