Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour,both term and preterm

The onset of labour in rodents and in humans is associated with physiological inflammation which is manifested by infiltration of activated maternal peripheral leukocytes (mPLs) into uterine tissues. Here, we used flow cytometry to immunophenotype mPLs throughout gestation and labour, both term and preterm. Peripheral blood was collected from non‐pregnant women and pregnant women in the 1st, 2nd and 3rd trimesters. Samples were also collected from women in active labour at term (TL) or preterm (PTL) and compared with women term not‐in‐labour (TNIL) and preterm not‐in‐labour (PTNIL). Different leukocyte populations were identified by surface markers such as CD45, CD14, CD15, CD3, CD4, CD8, CD19 and CD56. Their activation status was measured by the expression levels of CD11b, CD44, CD55, CD181 and CD192 proteins. Of all circulating CD45+ leukocytes, we detected significant increases in CD15+ granulocytes (i) in pregnant women versus non‐pregnant; (ii) in TL women versus TNIL and versus pregnant women in the 1st/2nd/3rd trimester; (iii) in PTL women versus PTNIL. TL was characterized by (iv) increased expressions of CD11b, CD55 and CD192 on granulocytes; (v) increased mean fluorescent intensity (MFI) of CD55 and CD192 on monocytes; (vi) increased CD44 MFI on CD3+ lymphocytes as compared to late gestation. In summary, we have identified sub‐populations of mPLs that are specifically activated in association with gestation (granulocytes) or with the onset of labour (granulocytes, monocytes and lymphocytes). Additionally, beta regression analysis created a set of reference values to rank this association between immune markers of pregnancy and to identify activation status with potential prognostic and diagnostic capability.     

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. withCryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyteand granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogenously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.     

Effect of the EM Bokashi® Multimicrobial Probiotic Preparation on the Non-specific Immune Response in Pigs

The aim of the study was to determine the effect of EM Bokashi® on the phagocytic activity of monocytes and granulocytes, oxidative burst, SWC3, and CD11b + CD18+ expression on monocytes and granulocytes, and the serum concentration of cytokine and lysozyme in pig. 60 Sixty female piglets were divided into two groups: I – control and II – experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® was added to the basal feed. Flow cytometry was used to determine selected non-specific immune response parameters, intracellular production of hydrogen peroxide by peripheral granulocytes and monocytes, and surface particles in peripheral blood. The EM Bokashi® preparation used in the study was found to increase phagocytic activity mainly in monocytes, with an increased percentage of phagocytic cells in the experimental group. The highest serum lysozyme concentration in the piglets in the experimental group (2.89 mg/dl), was noted on day 42 of the study. In the group of pigs receiving EM Bokashi®, the percentage of phagocytic cells with SWC3 (monocyte/granulocyte) expression was statistically significantly higher than in the control. The increase in the number of cells with SWC3 (monocyte/granulocyte) expression in the peripheral circulation in combination with the greater capacity of the cells for phagocytosis and respiratory burst confirms that the non-specific immune response was modulated in the pigs supplemented with EM Bokashi®.

     

Phenotypic and functional changes of circulating monocytes and polymorphonuclear leucocytes from elderly persons

The function and phenotype of monocytes and granulocytes in the elderly is consistently remodelled. Because leucocyte adhesion molecules play important roles in mediating a wide variety of leucocyte functions, age-related changes in their expression on granulocyte and monocyte surfaces could be partially responsible for immune dysfunctions during senescence. Considering the central role of innate immunity in the process of immunosenescence and the involvement of cell adhesion molecules (CAM) in the great majority of leucocyte functions, we studied the expression of CD50 and CD62L adhesion molecules in peripheral blood granulocytes and monocytes from healthy elderly and young subjects. We show here that the percentage of granulocytes and monocytes expressing CD62L is decreased in the elderly, whereas its density expression is unchanged on both cell types. A downregulation of the density expression of CD50 at a per cell level characterizes granulocytes in the elderly, whereas CD50 expression on monocytes from old subjects shows a peculiar attitude: its density expression decreases whereas the number of positive cells is expanded. The downregulation of this receptor on granulocytes from aged people could determine a state of hyperactivation contributing to the proinflammatory status of the elderly, while the lower expression on monocytes could therefore contribute to the impaired antigen presentation in the elderly. On the other hand, the increased number of CD50 positive monocytes in the elderly, despite its decreased density expression at a per cell level, could be interpreted as an attempt to counteract the inability to mount strong immune responses. Both CD50 and CD62L changes in ageing polymorphonuclear (PMN) cells allow recognition as non-self or senescent self to permit macrophages in the liver and spleen to remove them from the circulation. The increased proportion of granulocytes and monocytes lacking CD62L and the downregulation of CD50 intensity expression on both cell types may suggest a state of in vivo activation. Therefore, CD50 and CD62L shedding from the cell surface of activated granulocytes and monocytes could be interpreted as a tentative to counteract the dangerous effects of an excessive chronic inflammation in the elderly. However, the increased proportion of CD62L negative granulocytes in the elderly leads to an impairment in cell adhesion which is the first line of response to acute inflammatory stimuli. This phenomenon likely contributes to the increased susceptibility to acute infections of elderly people.     

Expression of adhesion and activation molecules on circulating monocytes in children with Helicobacter pylori infection

Objectives: The aim of this study was to assess the cell surface expression of adhesion (CD11a, CD11b, CD11c, CD18, CD54, and CD58) and activation (CD14, HLA‐DR, and CD16) molecules on the circulating monocytes in Helicobacter pylori (H. pylori)‐infected and noninfected children with gastritis, with the goal of comparing the results with those obtained from the controls. Materials and Methods: Ninety‐four children were studied: 47 of them with H. pylori infection (of those 25 children after the failure of eradication therapy) and 26 children with gastritis where H. pylori infection was excluded, as well as 21 controls. H. pylori infection status was assessed based on [¹³C] urea breath test, rapid urease test, and histology. Analysis of the monocyte surface molecule expression was carried out by flow cytometry. Results: H. pylori‐infected children and children who experienced a failure of the eradication therapy differed significantly in the expression of adhesion and activation molecule on circulating monocytes. A decrease, both in the proportion of CD11c‐ and CD14‐bearing monocytes, and the expression of CD11c and CD14 molecules on circulating monocytes, was found in children in whom the eradication therapy failed (p < .05). Low expression of CD11b (p = .04) and CD18 (p = .02) integrins on monocytes was also observed. Additionally, the percentage of HLA‐DR‐bearing monocytes was decreased (p = .04), while the CD16 density receptor was increased (p = .02). Compared with the controls, low percentage of CD16‐positive monocytes was noted in noninfected children with gastritis (p = .01). Conclusion: H. pylori eradication therapy in children causes inhibition of inflammatory response via a reduction in CD11b, CD11c, and CD18 beta2 integrin monocyte expression.     

Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45), and Fc gamma-RIII (CD16) upon stimulation of human peripheral leukocytes with human C5a.

Rapid increases in the membrane expression of C3 receptors on granulocytes and monocytes in response to the anaphylatoxin C5a have previously been described. In this study we demonstrate increases in the membrane expression of neutral endopeptidase (NEP, CD10, CALLA), aminopeptidase N (APN, CD13), tyrosine phosphatase (CD45/CD45Ro) and the Fc R Fc gamma-RIII (CD16) on granulocytes within minutes of treatment with human C5a. Monocytes responded to C5a with increases in CD13 and CD45/CD45Ro. These membrane modulations could be prevented by preincubating the C5a preparations with anti-C5a mAb C17/5 but not by pretreating the cells with cycloheximide. Increases of CD10, CD13, and CD11b but not CD11a (LFA-1) were also observed in leukocytes from patients undergoing hemodialysis with cuprophan membranes. The increase of CD16 on granulocytes was dependent on the presence of plasma during in vitro activation with C5a indicating that plasma contains inhibitors which prevent the previously described loss of Fc gamma-RIII upon stimulation of the cells.     

Expression of tumour necrosis factor receptors (CD120a and CD120b) on bronchoalveolar cells.

It is generally accepted that physiological modulators for tumour necrosis factor (TNF) are present in a variety of body fluids including serum. Among these modulators are soluble TNF receptors (TNF-R) that are cleaved from the extracellular domain of the TNF-Rs. Two receptors of different structures with molecular weights of 55 kDa (CD120a) and 75 kDa (CD120b) are known to be expressed on monocytes, lymphocytes, granulocytes and other cells of peripheral blood. The aim of our study was to determine the expression of CD120a and CD120b on bronchoalveolar lavage cells (BAL cells). BAL cells of 14 patients with different pulmonary disorders were stained with anti-CD120a and anti-CD120b monoclonal antibodies and were differentiated by FACS analysis. Both TNF-Rs are expressed on monocytes, macrophages, lymphocytes and granulocytes of the BAL. Although the relation of CD120a to CD120b is individual for a given cell type and an individual patient, strict correlations between both receptors were observed for BAL monocytes and alveolar macrophages. CD120a are expressed on 29.7% of alveolar macrophages; similar data were obtained for CD120b. 24.3% of the BAL monocytes were positive for CD120a and 25.5% for CD120b. 4.1% of the BAL lymphocytes were positive for CD120a whereas the percentage of CD120b positive BAL lymphocytes was approximately six times greater. Analysis of BAL granulocytes revealed 21.2% cells positive for CD120a and 11.6% for CD120b. In contrast to the BAL cells named above there was no positive correlation between CD120a and CD120b expression on BAL lymphocytes and granulocytes. We were able to show that TNF-Rs of BAL cells, like those of blood cells, are shedded in vitro after incubation with or without lipopolysaccharide (LPS), detected as TNFalpha-inhibitor activity in cell culture supernatant. In conclusion, BAL cells express and shed TNF-Rs, as is known for cells of other body compartments.     

Cariporide Counteracts Atherosclerosis‐related Functions in Monocytes from Obese and Normal Individuals

Objective: This study aimed to show the effect of high glucose concentrations in combination with a pharmaceutical analog of the Na⁺/H+ antiport inhibitor, cariporide, on scavenger receptor CD36 expression, cell adhesion, and cell migration of human monocytes derived from obese and normal individuals. Research Methods and Procedures: Monocytes were isolated from six healthy obese individuals and six healthy age‐ and sex‐matched controls by use of whole blood Percoll sedimentation and plastic surface monocyte binding. The density of CD36 scavenger receptors on the surface of monocytes was assessed by the use of a fluorescent fluorescein isothiocyanate (FITC)‐linked monoclonal antibody. Transmigration of monocytes through laminin‐1–coated filters was performed on 5‐μm pore Transwell culture inserts. Monocyte attachment to laminin was estimated by a solid phase assay. Results: High glucose concentrations caused an increase in monocytes from normal and obese individuals in the expression of CD36 receptors and positively influenced monocyte migration and adhesion to laminin. Cariporide together with glucose counteracted these effects. The effects of migration and adhesion of monocytes to laminin were specific to glucose, because the effect was significantly higher when monocytes were incubated in the presence of 20 mM of glucose than in the presence of 20 mM of fructose. Monocytes from obese subjects showed greater response than in normal to all of the studied effects, with the highest response in laminin attachment. Discussion: The data of this study suggest that cariporide counteracts atherosclerosis‐related functions through Na⁺/H+ antiport inhibition in monocytes from both normal and obese individuals.     

Altered chemokine receptor profile on circulating leukocytes in human heart failure

Chemokines and their receptors have been implicated in the pathogenesis of different forms of heart failure (HF). We examined CC-and CXC-chemokine receptor expression in fresh peripheral blood leukocyte populations from 24 end-stage HF patients consisting of coronary artery disease (CAD; n=6) and hypertrophic cardiomyopathy (HCM; n=7) or idiopathic dilated cardiomyopathy (IDCM; n=8) or valvular disease (VD; n=3) and compared the data with 18 healthy controls. Levels of CCR1, 2, 3, 4, 5, and 7, and CXCR1, 2, 3, and 4 were measured by flow cytometry, and the expression profile was assessed as molecules of equivalent soluble fluorochrome units as well as frequency (percentage) of CD3⁺, CD4⁺, and CD8⁺ T cells and monocytes or granulocytes. Frequency of CD3⁺ CXCR4⁺, CD3⁺ CXCR1⁺, and CD3⁺ CXCR3⁺ cells was significantly increased in HF patients, whereas only CCR7 and CXCR4 expression levels were elevated on CD3⁺ cells. Both CD4⁺ CXCR4⁺ and CD8⁺ CXCR4⁺ cell frequencies were significantly increased irrespective of cardiac disease etiology. Elevated CCR7 expression was less pronounced on CD4⁺ than CD8⁺ cells in patients with CAD and IDCM. Expression of CXCR4 on CD8⁺ cells was upregulated substantially, regardless of the cause of disease. CD8⁺ CXCR1⁺ and CD8⁺ CXCR3⁺ but not CD4⁺ CXCR1⁺ or CD4⁺ CXCR3⁺ cells were increased in the HF patients with IDCM and CAD, respectively. Expression of CXCR1 or CXCR3 on both CD4⁺ and CD8⁺ cells did not differ in all the groups. For monocytes, frequency of CD14⁺ CCR1⁺ and CD14⁺ CCR2⁺ cells was significantly decreased in CAD patients, whereas, increase in CD14⁺ CXCR4⁺ cell frequency was accompanied with elevated CXCR4 expression. On granulocytes, CXCR1 and CXCR2 receptors were downregulated in all patients, compared with controls. Our results suggest that the altered expression profile of CC- and CXC-chemokine receptors on circulating leukocyte populations involves enhanced activation of the immune system, perhaps as part of the pathogenic mechanisms in HF. Modulation of the chemokine network could offer interesting novel therapeutic modalities for end-stage HF.     

Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers

It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.     

Aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function

Chronic inflammation in older individuals is thought to contribute to inflammatory, age‐related diseases. Human monocytes are comprised of three subsets (classical, intermediate and nonclassical subsets), and despite being critical regulators of inflammation, the effect of age on the functionality of monocyte subsets remains to be fully defined. In a cross‐sectional study involving 91 healthy male (aged 20–84 years, median 52.4) and 55 female (aged 20–82 years, median 48.3) individuals, we found age was associated with an increased proportion of intermediate and nonclassical monocytes (P = 0.002 and 0.04, respectively) and altered phenotype of specific monocyte subsets (e.g. increased expression of CD11b and decreased expression of CD38, CD62L and CD115). Plasma levels of the innate immune activation markers CXCL10, neopterin (P < 0.001 for both) and sCD163 (P = 0.003) were significantly increased with age. Whilst similar age‐related changes were observed in both sexes, monocytes from women were phenotypically different to men [e.g. lower proportion of nonclassical monocytes (P = 0.002) and higher CD115 and CD62L but lower CD38 expression] and women exhibited higher levels of CXCL10 (P = 0.012) and sCD163 (P < 0.001) but lower sCD14 levels (P < 0.001). Monocytes from older individuals exhibit impaired phagocytosis (P < 0.05) but contain shortened telomeres (P < 0.001) and significantly higher intracellular levels of TNF both at baseline and following TLR4 stimulation (P < 0.05 for both), suggesting a dysregulation of monocyte function in the aged. These data show that aging is associated with chronic innate immune activation and significant changes in monocyte function, which may have implications for the development of age‐related diseases.     

Aged mice have increased inflammatory monocyte concentration and altered expression of cell-surface functional receptors

The expression of monocyte cell-surface receptors represents one index of immune dysfunction, which is common with aging. Although mouse models of aging are prevalent, monocyte subset assessment is rare. Our purpose was to compare cell receptor expression on classic (CD115⁺/Gr-1^high) and non-classic (CD115⁺/Gr-1^low) monocytes from 80- or 20-week-old CD-1 mice. Three-colour flow cytometry was used to determine the concentration of monocyte subsets and their respective cell-surface expression of TLR2, TLR4, CD80, CD86, MHC II and CD54. These receptors were selected because they have been previously associated with altered monocyte function. Data were analysed with independent t-tests; significance was set at P < 0.05. Old mice had a greater concentration of both classic (258%, P = 0.003) and non-classic (70%, P = 0.026) monocytes. The classic : non-classic monocyte ratio doubled in old as compared with that in young mice (P = 0.006), indicating a pro-inflammatory shift. TLR4 (↓27%, P = 0.001) and CD80 (↓37%, P = 0.004) were decreased on classic monocytes from old as compared with those from young mice. TLR2 (↑24%, P = 0.002) and MHCII (↓21%, P = 0.026) were altered on non-classic monocytes from old as compared with those from young mice. The increased classic : non-classic monocyte ratio combined with changes in the cell-surface receptor expression on both monocyte subsets is indicative of immune dysfunction, which may increase age-associated disease risk.     

Integrin expression and H2O2 production in circulating and splenic leukocytes of obese rats

Objective: Obesity is associated with oxidative stress and inflammation. We hypothesized that the pro‐inflammatory state in obesity may result in spontaneous activation and, hence, increased generation of reactive oxygen species (ROS) and integrin expression in the circulating leukocytes. Methods: Flow cytometry was used to determine integrin expression (immunostaining) as well as superoxide and hydrogen peroxide productions (fluorescent probes) in the peripheral blood and splenic leukocyte of 24‐week‐old male obese normotensive and not‐as‐yet diabetic Zucker rats (n = 6) and their lean counterparts (n = 6). Results: Obese rats had hyperlipidemia and normal arterial pressure, plasma glucose, and creatinine concentrations. Nevertheless, obese rats exhibited increased hydrogen peroxide production by circulating and splenic CD4+ and CD8+ T lymphocytes and by splenic macrophages. This was accompanied by up‐regulations of CD11a expression in the peripheral blood and splenic CD4+ T cells, CD11b in circulating macrophages, and CD11a and CD18 in circulating granulocytes. Conclusion: The study revealed direct evidence of spontaneous leukocyte activation and increased ROS generation by T lymphocytes and monocytes in the peripheral blood of obese Zucker rats before the development of diabetes or hypertension. These findings illustrate the link between obesity, oxidative stress, and inflammation.     

Down-regulation of HLA class II and costimulatory CD86/B7-2 on circulating monocytes from melanoma patients

Antigen-presenting cells are crucial for the induction of an antigen-specific antitumoral immune response. Deteriorations in the expression pattern of cell surface molecules important for the presentation of antigens might therefore be indicative of an impaired immune response status in cancer patients. In the present study we investigated the expression of MHC class I and class II molecules, of the costimulatory molecules CD80/B7-1 and CD86/B7-2, of the adhesion molecule CD11c, and of the marker of activation CD71 on CD14⁺ peripheral blood monocytes (PBMs) from 144 melanoma patients in different stages of disease and 43 healthy controls, by flow cytometric analysis. We found a decreased expression of HLA-DR (p<0.0005), HLA-DQ (p=0.006), HLA-DP (p<0.0005), and CD86/B7-2 (p=0.001) on PBMs from melanoma patients compared with healthy controls, whereas no significant difference could be detected in the expression of HLA class I antigens and CD80/B7-1. This down-regulated expression was associated with disease progression. In contrast, CD71 expression was stage-dependently increased on PBMs from melanoma patients compared with healthy controls (p=0.024). No correlation was found between the PBM surface expression pattern and age, gender, tumor load, and current mode of therapy of the patients. The observed down-regulation of HLA class II and CD86/B7-2 on melanoma patients PBMs might reflect an ineffective antigen-presenting function contributing to an impaired antigen-specific immune response in these patients.Both authors S. Ugurel and D. Uhlig contributed equally to this work     

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.     

Resident cardiac immune cells and expression of the ectonucleotidase enzymes CD39 and CD73 after ischemic injury

Background

The ectoenzymes CD39 and CD73 are expressed by a broad range of immune cells and promote the extracellular degradation of nucleotides to anti-inflammatory adenosine. This study explored the abundance of CD73 and CD39 on circulating and resident cardiac leukocytes and coronary endothelial cells under control conditions and in response to inflammation following myocardial ischemia and reperfusion (I/R).

Methods and Results

A method was elaborated to permit FACS analysis of non-myocardial cells (resident leukocytes, coronary endothelium and CD31⁻ CD45⁻ cells) of the unstressed heart. Under control conditions the murine heart contained 2.3×10³ resident leukocytes/mg tissue, the most prominent fraction being antigen-presenting mononuclear cells (CD11b⁺ CD11c⁺ F4/80⁺ MHCII⁺) followed by B-cells, monocytes and T-cells. CD73 was highly expressed on circulating and resident cardiac lymphoid cells with little expression on myeloid cells, while the opposite was true for CD39. Cardiomyocytes and erythrocytes do not measurably express CD39/CD73 and CD39 dominates on coronary endothelium. Three days after I/R, CD73 was significantly upregulated on invading granulocytes (2.8-fold) and T-cells (1.5-fold). Compared with coronary endothelial cells, CD73 associated with leukocytes comprised 2/3 of the total cardiac CD73.

Conclusion

Our study suggests that extracellular ATP formed during I/R is preferentially degraded by CD39 present on myeloid cells, while the formation of immunosuppressive adenosine is mainly catalysed by CD73 present on granulocytes and lymphoid cells. Upregulated CD73 on granulocytes and T-cells infiltrating the injured heart is consistent with the existence of an autocrine adenosinergic loop which may promote the healing process.     

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals

Abstract In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794–28725, CD11b/CD18: 5300–11939 and L-selectin: 35662–61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and “normals” must be interpreted with caution.     

Staphylococcal lipase affects phagocytosis of Staphylococcus aureus by human granulocytes and monocytes.

The rate of immunological and non-immunological phagocytosis of staphylococci by lipase pre-treated human granulocytes and monocytes was compared. It was found that the effect of this enzyme on two types of cells is opposite. Lipase decreases phagocytosis by granulocytes and increases by monocytes. The revealed differences between phagocytosing cells studied prompted us to investigate the influence of lipase on Fc receptors on these cells (rosette EA test). The different susceptibility of Fc receptors on non-activated phagocytes to lipase was found. This could be at least partially responsible for the difference observed between phagocytic activity of granulocytes (decreased) and monocytes (increased) pretreated with staphylococcal lipase. Inactivated enzyme showed a similar effect as active enzyme in the case of granulocytes. However, inactivated enzyme had no effect on rosette formation by lipase pretreated monocytes, indicating an enzymatic effect.     

Selective gene expression and activation-dependent regulation of vasoactive intestinal peptide receptor type 1 and type 2 in human T cells

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4(+) T cells and CD8(+) T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4(+) T cells than in CD8(+) T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4(+) T cells and CD8(+) T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4(+) T cell subpopulations is highly correlated with T cell activation.