Functional and phenotypic changes in monocytes from patients with tuberculosis are reversed with treatment

Alterations of monocyte/macrophages have been reported in patients with tuberculosis (TB), but their significance is poorly understood. Blood mononuclear cells from patients with different clinical forms of TB, at various times of anti-TB treatment, and healthy tuberculin positive individuals, were double-stained for CD14 plus CD206, TLR-2, IFN-gammaR1, CD40, HLA-DR, CD36 and CD163, and analyzed by flow cytometry. Monocytes were infected with Mycobacterium tuberculosis H37Rv and 24h later the phenotype, induction of necrosis and apoptosis and production of tumor necrosis factor TNFalpha, interleukin (IL)-10 and IL-12p40 were determined. TB patients presented higher percentage of CD14+ cells but lower percentage of CD14+DR+ and CD14+CD36+ cells. Expression of CD14, HLA-DR and CD36 was decreased in TB patients. Normal percentages and expression were restored during anti-TB treatment. Monocytes from TB patients underwent necrosis and apoptosis after M. tuberculosis infection, whereas monocytes from healthy controls exhibited only apoptosis. Anti-TB treatment reverted necrosis. There were no differences between the various clinical forms of TB. In vitro M. tuberculosis infection decreased expression of the membrane molecules studied. HLA-DR and CD36 inhibition correlated with induction of apoptosis. Restoration of monocyte alterations during anti-TB treatment suggests that such alterations may be caused by the high M. tuberculosis load present during active disease.     

Human blood dendritic cell-like B cells isolated by the 5G9 monoclonal antibody reactive with a novel 220-kDa antigen.

We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40-60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either kappa- or lambda-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9- PBMC, 5G9- B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.     

In vivo radioprotection by 5-androstenediol: stimulation of the innate immune system

We showed previously that 5-androstenediol stimulates myelopoiesis, increases the numbers of circulating neutrophils and platelets, and enhances resistance to infection in gamma-irradiated mice. We have extended those studies to include monocytes, natural killer (NK) cells, eosinophils and basophils, and we have measured the activation marker CD11b using flow cytometry. Androstenediol (160 mg/kg) was administered subcutaneously to female B6D2F1 mice 24 h before whole-body gamma irradiation. Androstenediol treatments increased the blood levels of neutrophils, monocytes and NK cells in unirradiated animals; decreased the numbers of circulating eosinophils; and ameliorated radiation-induced decreases in neutrophils, monocytes, NK cells, erythrocytes and platelets. The androstenediol treatments had no significant effect on the numbers of circulating B cells or T cells. CD11b labeling intensity on monocytes was decreased slightly after androstenediol treatment. In contrast, radiation or androstenediol alone caused increases in CD11b labeling intensity on NK cells. Androstenediol and radiation combined caused a marked increase in NK cell CD11b. The results indicate that androstenediol increases the numbers of the three major cell types of the innate immune system (neutrophils, monocytes and NK cells), that androstenediol-induced changes in blood elements in irradiated animals persist for at least several weeks, and that there is a significant positive interaction between radiation and administration of androstenediol in the activation of NK cells.     

优化CD115稳定性因素以精确检测小鼠单核细胞及其亚群

单核细胞是机体防御系统的重要组成部分,单核细胞数量发生改变一定程度上可反映机体炎症或其他疾病状态。应用流式细胞术对单核细胞表面抗原进行检测,可了解单核细胞占比变化,有助于揭示单核细胞在疾病发生和转归中的作用。鉴于小鼠单核细胞特异性抗原CD115的稳定性较差,在流式检测过程中,对其影响因素进行优化,以确保检测结果的准确性。首先,探讨CD115抗原-抗体结合前后温度和放置时间对其影响及多聚甲醛（paraformalclehyde,PFA）固定对CD115稳定性的保持作用。在此基础上,通过组合流式抗体CD11c、 CD49b、 Ly6G、 Ter119、 CD3e 和 B220选定阴性细胞群,再用CD11b和CD115圈门得到双阳性的单核细胞,最后根据Ly6C表达强弱区分单细胞亚群。结果显示,温度和放置时间对小鼠单核细胞CD115的稳定性影响显著。将小鼠样本在冰上放置6 h时,CD11b和CD115双阳性细胞比例仍保持稳定,在室温放置1 h即明显下降,在37 ℃放置0.5 h则大部分消失。该结果在抗体结合前后趋势一致。PFA固定后,在4 ℃条件下避光保存, CD11b和CD115双阳性细胞比例可稳定保持3 d。在此优化条件下,应用组合抗体可较好地分析小鼠单核细胞及其亚群,为精准检测小鼠单核细胞提供了技术支持,并为进一步研究单核细胞生物学特性及其在疾病中的作用提供了可能。     

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.     

Treatment with recombinant IFN-gamma decreases cell surface CD4 levels on peripheral blood monocytes and on myelomonocyte cell lines

The human cell surface protein CD4 is not only an important accessory molecule in the activation of MHC class-II-restricted T cells, but has also been implicated to be a receptor for the human immunodeficiency virus HIV-I on lymphoid and monocytic cells. We have found that a 24-h treatment of the promonocytic leukemia cell line U937 with rIFN-gamma decreases the expression of the CD4 Ag by 50% as measured by cytofluorographic analysis. The decrease in CD4 expression was dependent on the concentration of rIFN-gamma, with maximal effects occurring at 20 to 200 U/ml. The decrease appeared to be due to actual loss of the CD4 molecule from the cell surface rather than masking of a particular epitope, inasmuch as similar results were obtained with the OKT4 and OKT4A antibodies. The effect of rIFN-gamma to decrease CD4 expression was not due to a general loss of cell surface Ag, because the binding of OKM1 and anti-HLe-1 increased after rIFN-gamma treatment. Treatment of rIFN-gamma also decreased cell surface CD4 expression on the promyelocytic leukemia cell line HL-60, and on the monocytic cell line THP-1, although the extent of the decrease was less than on U937 cells. Freshly isolated normal peripheral blood monocytes treated for 48 h with rIFN-gamma bound much less OKT4 or OKT4A antibody than cells incubated in the absence of rIFN-gamma. Moreover, treatment with rIFN-gamma reduced the percentage of peripheral blood monocytes that were positive for the CD4 Ag. In contrast with the decrease in CD4 levels on rIFN-gamma-treated monocytes, treatment with rIFN-gamma had no effect on CD4 levels on peripheral blood T lymphocytes or T cell lines.     

Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 µg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14⁺⁺CD16⁺ cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14⁺⁺CD16⁺ monocyte subset.     

Regulation of human monocyte cell-surface and soluble CD23 (Fc epsilon RII) by granulocyte-macrophage colony-stimulating factor and IL-3.

We have investigated the regulation of expression of cell-surface and soluble CD23 (sCD23) by purified human peripheral blood monocytes and in cultures of human whole blood. IL-3, IL-4, and GM-CSF were found to markedly enhance the expression of CD23 on the surface of elutriated monocytes and to increase levels of sCD23 in monocyte-culture supernatants. The induction of CD23 expression by monocytes was confirmed at the mRNA level by Northern blot analysis. The ability of GM-CSF, IL-3, or IL-4 to induce cell-surface CD23 on monocytes was inhibited by specific neutralizing antibodies to the corresponding cytokine. IL-3 and GM-CSF induced maximal surface CD23 expression on monocytes by 24 to 48 h, followed by a slight decline at 72 and 96 h. In contrast, IL-4 induced a progressive increase in monocyte CD23 expression that reached a maximum at approximately 72 h. IL-4, GM-CSF, and IFN-gamma increased both surface and soluble CD23 expression by the monocytic cell line U937, whereas IL-3 had no effect. The plasma from fresh human whole blood or nonstimulated whole blood cultured for 24 to 48 h contained detectable sCD23, and addition of IL-3, IL-4, or GM-CSF to these cultures resulted in increased levels of this molecule. Two-color flow cytometry revealed that IL-3, but not GM-CSF, also enhanced CD23 expression by B cells enriched from PBMC, although the effect of IL-3 was weak in comparison with that of IL-4. These findings may have important implications for the in vivo therapeutic use of these cytokines.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Cellular origin of histamine-releasing factor produced by peripheral blood mononuclear cells

Histamine releasing factors (HRF) are a group of cytokines that release histamine and other mediators from mast cells and basophils. It has been speculated that HRF might play a major role in the pathogenesis of allergic diseases. Most investigators have studied PBMC as a source of HRF. This study was undertaken to investigate the cellular origin of HRF. Peripheral blood was processed to isolate and purify monocytes, T cells, CD4- T cells, CD8- T cells and B cells by using plastic adherence, 2-aminoethylisothiomonium-treated SRBC rosetting and negative selection with the use of mAb OKM1, OKT11, OKT8, OKT4, and OKB7 plus C. Highly purified subpopulations of PBMC were cultured alone or in the presence of Con A for 24 h. Supernatants were harvested, dialyzed, and assayed for HRF activity in the basophil histamine release test. We found that all subpopulations of PBMC including T cells, CD4- T cells, CD8- T cells, B cells, and monocytes produce variable quantities of HRF. The spontaneous production is very high in B cells but only barely measurable in T cells and monocytes. The synthesis of HRF by B cells was confirmed by abolishing the release of the activity after treatment of B cells with OKB7 mAb and C. Stimulation of cell populations by Con A significantly enhances HRF production by PBMC and T cells but not by B cells and monocytes. In mixing experiments, unstimulated monocytes + B cells showed synergism, but other combinations demonstrated an additive effect. This is the first demonstration of HRF production by human peripheral blood B cells. The results of this study also suggest that histamine releasing cytokines are of multiple cellular origin. This perhaps contributes to their molecular heterogeneity.     

Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes

CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14.     

Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis

Background

Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14⁺ monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis.

Aim

To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.

Methods

CD14⁺ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.

Results

GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.

Conclusion

SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties.     

Increased liver temperature efficiently augments human cellular immune response: T-cell activation and possible monocyte translocation

Hyperthermia (HT), in combination with other conventional therapeutic modalities, has become a promising approach in cancer therapy. In addition to heat-induced apoptosis, an augmented immunological effect is considered to be a benefit of hyperthermic treatment over chemo- or radiotherapy. Here, we investigated the effect of regional HT targeting the liver on immune cells, especially T cells and antigen-presenting cells, which are important in recognizing and eliminating tumor cells and pathogens such as viruses. In healthy volunteers exposed to such regional HT, both CD4⁺ and CD8⁺ T cells that express an activation marker CD69 increased transiently at 1 h post-treatment, with a subsequent decrease to base levels at 6 h after the treatment. At 24 h post-treatment, the percentage of CD69-positive cells significantly increased again but only among CD8⁺ T cells. IFN-γ production from PHA-stimulated peripheral blood mononuclear cells was gradually and significantly increased in the 2 days following the heating procedure, peaking at 36 h post-treatment. Furthermore, we found marked increases in plasma levels of IL-1β and IL-6 starting at 24 h post-treatment. With regard to the number of each leukocyte subpopulation, a transient and dramatic decrease in the number of a subset of monocytes, CD14⁺ CD16⁻ cells, was observed at 1 h after the hyperthermic treatment, suggesting that the regional HT aimed at the liver may have influenced the extravasation of blood monocytes. No significant changes in T-cell activities or monocyte counts were observed in the volunteers exposed to heating of the lungs or the legs. These results suggest that heating of the liver may efficiently induce cellular immune responses to liver cancers.     

Effects of hydroxyurea on monoclonal antibody production induced by anti-mIgG and LPS stimulation on murine B cell hybridomas

Chemical treatment with hydroxyurea (HU) has been selected as a simple and low cost strategy to generate a cell population enriched for the G1 phase. After the chemical treatment with HU, cells were stimulated with anti-mIgG to test if the positive effects of anti-mIgG on CD40 expression and specific IgG2a production rate were improved upon a cell population with a higher percentage of cells in G1 phase at the beginning of the cell culture. In addition, other treatments assayed in this work were the cell stimulation with Lipopolysaccharide (LPS) both before and after the HU treatment. It has been observed that the use of HU under conditions able to maintain the cells in viable state (0.1 mM for 20 h), has a negative effect on CD40 expression and specific IgG2a production rate induced by anti-mIgG. The positive effect of LPS on cell stimulation induced by anti-mIgG is reduced on cells treated with HU.     

Differential Migration and Activation Profile of Monocytes after Trophoblast Interaction

Macrophages at the maternal-placental interface coordinate opposite demands under the control of trophoblast cells such as the response against pathogens on one hand, and apoptotic cell clearance and wound healing with the production of suppressor cytokines. Here, we investigated whether trophoblast cells induce maternal monocyte activation towards an alternative activated macrophage profile and whether bacterial or viral stimuli modulate their migratory properties. We used an in vitro model of the maternal-placental interface represented by co-cultures of CD14+ cells isolated from fertile women with first trimester trophoblast cell line (Swan-71 cells) in the presence or absence of pathogen associated molecular pattern (PAMP) stimuli lipopolysaccharide (LPS), peptidoglycan (PGN) or poly [I:C]). Maternal CD14+ cells showed increased CD16 and CD39 expression, both markers associated to an alternative activation profile, with no changes in CD80 expression after trophoblast cell interaction. These changes were accompanied by increased IL-10 and decreased IL-12 production by CD14+ cells. After stimulation with LPS, PGN or poly [I:C], monocytes co-cultured with trophoblast cells had lower production of TNF-α and IL-1β compared with non co-cultured monocytes. Interestingly, monocyte migration towards trophoblast cells was prevented in the presence of LPS or PGN but not after 24h of stimulation with poly [I:C]. LPS or PGN also decreased CCR5, CXCL-8 and CCL5 expression. Finally, trophoblast cells co-cultured with monocytes in the presence of pathological stimuli failed to increase chemokine expression, indicating a bidirectional effect. In conclusion, trophoblast might ‘instruct’ maternal monocytes to express an alternative activation profile and restrain their early recruitment under pathological threats as one of the first strategies to avoid potential tissue damage at the maternal-placental interface.     

Expression of complement regulatory proteins [membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59)] in endometrial stressed cells

In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.     

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma

Background

Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied.

Methods

We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry.

Results

Subjects with severe asthma had higher PAR-2 expression on CD14⁺⁺CD16⁺ monocytes (intermediate monocytes) and also higher percentage of CD14⁺⁺CD16⁺PAR-2⁺ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14⁺⁺CD16⁺PAR-2⁺ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14⁺⁺CD16⁺ PAR-2⁺ cells in peripheral blood compared to subjects without exacerbations.

Conclusions

PAR-2 expression is increased on CD14⁺⁺CD16⁺ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14⁺⁺CD16⁺ monocytes may play a role in the pathogenesis of severe asthma.     

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14(hi) /CD16(neg) , CD14(hi) /CD16(pos) and CD14(lo) /CD16(pos) ) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi) /CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo) /CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi) /CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies. ? 2012 International Society for Advancement of Cytometry.     

Moraxella catarrhalis lipooligosaccharide selectively upregulates ICAM-1 expression on human monocytes and stimulates adjacent naïve monocytes to produce TNF-alpha through cellular cross-talk

To elucidate the role of Moraxella catarrhalis lipooligosaccharide (LOS) in otitis media with effusion (OME), the effects of LOS on adhesion antigens of human monocytes were investigated. M. catarrhalis LOS selectively enhanced intercellular adhesion molecule 1 (ICAM-1 or CD54) expression on human monocytes by significantly increasing both the surface expression intensity and the percentage of ICAM-1⁺ cells. ICAM-1 upregulation on human monocytes by the LOS required surface CD14, TLR4, NF-κB p65 and c-Jun N-terminal kinase (JNK) activity. Our study also revealed that the LOS-induced surface ICAM-1 expression was partially mediated through a TNF-α dependent autocrine mechanism and could be further augmented by lipopolysaccharide-binding protein in serum. In addition, M. catarrhalis LOS also stimulated human monocytes to produce pro-inflammatory cytokines in both TLR4- and CD14-dependent pathways. Our results also indicated that enhanced surface ICAM-1 expression on monocytes may hinder their adherence to the lung epithelial monolayer. Furthermore, the LOS-activated human monocytes secreted a significantly high level of IL-8, and could stimulate adjacent naïve monocytes to produce TNF-α which was partially mediated via membrane ICAM-1 and IL-8/IL-8RA. These results suggest that M. catarrhalis LOS could induce excessive middle ear inflammation through a cellular cross-talk mechanism during OME.