Preparation and characterization of monoclonal antibodies recognizing two distinct differentiation antigens (Pro-Im1, Pro-Im2) on early hematopoietic cells

A series of monoclonal antibodies recognizing myeloid differentiation antigens were prepared by immunizing Balb/c mice with HL-60 cells. Hybrids secreting antibodies reactive with HL-60 cells but unreactive with peripheral blood mononuclear cells were isolated and further cloned. One clone was found to produce an IgG2a antibody recognizing an 85,000-dalton molecular weight surface glycoprotein, and a second clone was found to produce an IgM antibody recognizing a heat-stable determinant present on a glycolipid. We have termed these antigens Pro- Im1 and Pro-Im2, respectively (Pro for using HL-60 promyelocytes as an immunogen and Im for the presence of these antigens on immature cells). alpha Pro-Im1 and alpha Pro-Im2 were used to investigate the surface expression and tissue distribution of these two antigens. Pro-Im1 and Pro-Im2 were found to be brightly expressed on a fraction of fetal liver hematopoietic and bone marrow cells. Both antibodies mediated complement-dependent inhibition of CFU-GM, BFU-E, and CFU-GEMM formation assayed by soft agar colony and burst formation, indicating the expression of these antigens by early hematopoietic precursor cells. This was further confirmed by the induction of HL-60 cells by TPA to differentiate into more mature monocytes and macrophages, accompanied by the loss of both antigens. Pro-Im1 and Pro-Im2 were absent from peripheral blood monocytes, erythrocytes, and platelets, but Pro-Im2 was expressed on granulocytes. Both antigens were absent from thymocytes and peripheral T cells. Cytofluorographic analysis suggested their absence from peripheral blood B cells but that both were expressed on a minority of tissue B cells. Analysis of 150 cases of various myeloid and lymphoid malignancies demonstrated Pro-Im1 and Pro-Im2 expression on myeloblasts and promyelocytes from some acute myelogenous leukemias as well as some B cell malignancies, suggesting that these antigens are shared by early hematopoietic cells and a subset of B cells.     

The modulated expression of Mo5, a human myelomonocytic plasma membrane antigen

Mo5 is a 94-kd protein antigen expressed by human peripheral blood monocytes, neutrophils, and by all bone marrow myeloperoxidase-positive myeloid precursors (promyelocytes, myelocytes, metamyelocytes, and bands). Mo5 is borne by the malignant cells of 74% of patients (N = 27) with acute monocytic leukemia (French-American-British [FAB] group M4, M5), and 50% of patients (N = 38) with acute granulocytic leukemia (FAB M1, M2, and M3). Nonmyeloid cells in peripheral blood and bone marrow are Mo5-negative. The surface expression of Mo5 by myeloid cells is modulated by several experimental conditions: Exposure of neutrophils to calcium ionophore (1 mumol/L, 37 degrees C, ten minutes) under conditions resulting in degranulation of specific granules produces a three- to fourfold increase in the plasma membrane density of Mo5 antigen. This suggests that, in neutrophils, there is an intracellular pool of Mo5 antigen, which may be associated with specific granules, and that granule-associated Mo5 is translocated to the plasma membrane upon degranulation. Conversely, incubation of monocytes, neutrophils, U- 937, and Mo5-positive leukemia cells in medium containing anti-Mo5 monoclonal antibody results in a significant decrease in surface Mo5 expression. This loss of surface Mo5 is a rapid, temperature-dependent process (occurring within 30 minutes at 37 degrees C) that is produced by divalent anti-Mo5 immunoglobulin [F(ab')2 but not F(ab)]. After down- modulation, Mo5 is reexpressed by monocytes within 48 hours. Mo5 is therefore a human myelomonocytic differentiation antigen whose expression is modulated up or down depending on the nature of extracellular stimuli.     

Conversion of Cultured Monocytes/Macrophages into Endothelial-Like Cells through Direct Contact with Endothelial Cells

When culturing human umbilical vein endothelial cells in a culture medium containing 4% human serum albumin, it was possible to maintain the epithelioid morphology and function for several months without subculturing. When coculturing endothelial cells and labeled monocytes/macrophages (Mo/Phi) that were collected from peripheral blood and allowed to engulf fluorescent latex beads, some Mo/Phi changed their shapes and became epithelioid cells that were indistinguishable from vascular endothelial cells. This transformation started within several hours of coculturing. At 7 days after the start of coculturing, more than half of the labeled cells were identified as endothelial-like cells morphologically. Furthermore, morphologically altered Mo/Phi did not express Mo/Phi-specific antigens, ie, the MHC Class II molecule and CD68, but expressed VE cadherin and vWF, which are specific antigens for endothelial cells, and labeled cells that changed into endothelial-like cells no longer engulfed fluorescent latex beads. This strongly suggests that peripheral blood monocytes differentiate into endothelial-like cells.     

Fluorescent double labeling of normal and malignant hematopoietic cells by monoclonal antibodies (FITC) and anthracycline cytostatic drug (Daunomycin): a cytometric technique for analysis of drug uptake in hematopoietic cell subpopulations

A technique of simultaneous double labeling of normal and neoplastic hematopoietic cells with FITC-conjugated monoclonal antibodies directed to selectively expressed hematopoietic cell surface antigens (green fluorescence) and the anthracycline cytostatic drug (Daunomycin, red fluorescence) was described. Flow cytometric analysis of double labeled cells permitted anthracycline cell content determination in peripheral blood lymphocytes, granulocytes, monocytes from healthy donors, T- (MOLT-4), non-T, non-B (REH) and myelomonocytic (U-937) leukemic cell lines. After mixing peripheral blood lymphocytes from healthy individuals with cultured leukemic cells labeled on a restrictively expressed hematopoietic cell differentiation antigen (CALLA-CD10-, MHC class II-DR-antigen, a myelomonocytic differentiation antigen) detected by corresponding monoclonal antibodies (DGH-10-1-A9,Bra30, BraC8), the described technique allowed separate measurements of anthracycline content in leukemic cells vs. peripheral blood lymphocytes from healthy donors. Potential diagnostic aspects and research utilization of this technique are discussed.     

Human macrophage maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens

We have analyzed the expression of late differentiation antigens during terminal in vitro maturation of human macrophages (M phi) from blood monocytes (MO) in comparison to their distribution among mature M phi residing in various tissue sites. By immunizing mice with M phi derived from blood MO by culture on hydrophobic Teflon foils, monoclonal antibodies (mAbs) were developed (MAX.1, MAX.2, MAX.3, MAX.11) that reacted with lineage-restricted differentiation antigens. These antigens were expressed exclusively on M phi or were markedly increased after in vitro differentiation. The only overlap to another hemopoietic cell lineage was observed with MAX.3, which is shared by platelets and megakaryocytes. In the course of M phi maturation in vitro, the MAX.1 and MAX.3 antigens are detected within the cytoplasm two days before they appear on the cell surface. In contrast, the MAX.11 antigen is expressed simultaneously in the cytoplasm and at the cell surface, is found in varying degrees on a minor portion of blood MO and U937 cells, and is expressed rapidly at high density during early M phi differentiation in vitro. Among conventional mAbs that do not react with MO we found those against the transferrin (TF)-receptor, the BA-2, and the PCA1 antigen to label M phi. M phi matured in vivo and isolated from body fluids were positive with some but not all MAX mAbs. Distinctive patterns were observed with pulmonary M phi, exudate M phi from pleural and peritoneal effusions, synovial fluids, and early lactation milk. M phi from the alveolar space, for example, constantly expressed the MAX.2 antigen but not the MAX.3 antigen. Pleural effusion M phi, however, did not react with the MAX.1 mAb, but in most cases, it did react with the MAX.3 mAb. The detection of novel differentiation antigens, all expressed on monocyte-derived M phi but differently expressed on site-specific M phi in situ, underlines the remarkable heterogeneity among human M phi. The expression of these antigens is flexible because those MAX antigens that were not expressed in situ could be induced if cells from distinct tissue sites were cultured in vitro for several days. MAX mAbs may be of potential value to study both the sequential stages of maturation within the M phi lineage as well as differential developments induced by various culture conditions in parallel to environmental factors in vivo.     

Marker analysis of cloned populations of human monocytes

The presence of myelomonocytic progenitor cells in human peripheral blood was used for the analysis of cloned populations of human monocytes. Colonies of granulocytes and macrophages were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). Following 20-25 days of incubation, most colonies were found to consist of cells with monocyte-macrophage morphology. Cloned populations of monocytes were tested for several monocyte membrane markers and compared to noncloned adherent monocytes. HLA-DR, 63D3, LeuM2 antigens and Fc receptors were expressed on cells from individual colonies in similar proportions to their expression on noncloned monocytes. Some colonies were uniform in their negative expression of the 63D3 antigen, as were the noncloned monocytes. Although the clonality of cells tested was not directly proven, these results indicated that at least for some monocyte markers, heterogeneous expression was obtained in monoclonal populations of monocytes. It is possible, however, that testing of additional markers and functions may reveal homogeneous clones of monocytes and suggest the existence of stable subsets.     

Release of galactosyltransferase from human platelets and a subset of monocytes in culture

We show that human monocytes and platelets release considerable amounts of galactosyltransferase (GT) in serum-free culture as measured by the amount of incorporation of 3H-galactose into ovalbumin. Enzyme production was the greatest among medium-sized mononuclear cells separated by counter-current elutriation. The cells were adherent and positive for the monocyte-specific monoclonal antibody FMC-32. The activity in the monocyte fractions was not due to platelet contamination as shown from experiments in which platelets or platelet antigens were eliminated. Cell viability decreased by less than 3% during the overnight culture, and results from cell disruption experiments showed that the enzyme was not released from dead or dying cells. Cycloheximide inhibited release during 20 hours culture. Approximately 50% of the enzyme in the cell culture supernatant was pelletable at 105,000 g. Platelets released the enzyme more rapidly than did monocytes and were readily stimulated by thrombin to release more GT. Thrombin also increased monocyte GT activity after overnight incubation, but other stimulants, zymosan and lipopolysaccharide (LPS), decreased release. We conclude that GT is released into culture supernatants by platelets and by a subset of peripheral blood monocytes. These sources may account for a significant proportion of the serum enzyme and may be important in modification of extracellular carbohydrates during inflammation and coagulation.     

Characterization of a series of monoclonal antibodies against human T cells

A set of 6 monoclonal antibodies is characterized reacting only with human T-cells but not with B lymphocytes. The antibody BL-T1 binds to the E receptor, being there on all mature T cells and on the majority of thymocytes. All respectively the majority of peripheral T cells but only subpopulations of thymocytes are recognized by the monoclonals BL-T2, BL-T3, BL-T4 and BL-T5. For identification and quantification of mature human T cells especially the antibody BL-T2 is suitable. The antigen recognized by BL-E1 is missing on peripheral B lymphocytes but expressed not only on T cells but also on erythrocytes, monocytes and a fraction of granulocytes. The monoclonals discussed are compared with the monoclonals for identification of leucocyte differentiation antigens (cluster of differentiation) characterized on the 1st Int. Workshop on Human Leucocyte Differentiation Antigens (Paris, 1982).     

Arachidonic acid metabolites produced by platelet-depleted human blood monocytes: a possible role in thrombogenesis

The arachidonic acid metabolites produced by human peripheral blood monocytes were studied to determine which metabolites could have a role in thrombogenesis. Monocytes were found to be free of platelets by scanning electron microscopy and by measurement of 12-HETE. Human peripheral blood monocytes produce thromboxane as their major metabolite. Thromboxane levels reached a plateau at 12-16 hours of culture. Monocytes produced relatively little prostaglandin E2 or F2. In contrast to our control platelet preparation, neither A23187 (1-10 microM) nor exogenous arachidonic acid (0-40 microM) caused an increase in monocyte thromboxane B2. On the other hand, lipopolysaccharide (20 micrograms per ml), collagen (2.5 mg per 10(7) cells), and thrombin (5-10 units per ml) caused a two- to fivefold increase in monocyte thromboxane B2 in most donors but had no effect on prostaglandin F1 alpha levels. Blockage of thromboxane synthase by 1-benzylimidazole abolished thromboxane B2 production but did not increase prostaglandin F1 alpha. Finally, aspirin-treated platelets from a volunteer donor, which were refractory to 30 microM arachidonate, could be aggregated by isolated blood monocytes. Our data indicate that monocytes are capable of producing thromboxane in large amounts. The regulation of this increase, however, appears to be quite different from platelets. We postulate that monocytes may have a role in hemostasis by virtue of their ability to adhere at sites of vascular injury and release thromboxane, which may enhance platelet aggregation and thrombus formation.     

Initial characterization of monoclonal antibodies against human monocytes.

Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.     

Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells

Protease-activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain, unmasking a new N-terminus acting as tethered ligand. Whereas the role of PARs in platelets is well known, their presence and function in human monocytes and other antigen-presenting cells has not been characterized. Here it is demonstrated that human peripheral monocytes and monocyte-derived macrophages and dendritic cells differentially express PARs. Human monocytes express mainly PAR1 and less PAR3. Differentiation of monocytes into macrophages by either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) elicits enhanced expression of PAR1, PAR2, and PAR3. In contrast, dendritic cells differentiated from monocytes by GM-CSF and interleukin-4 (IL-4) strongly down-regulated PAR1, PAR2, and PAR3, both at the mRNA and the protein level. Down-regulation of the PAR expression was apparently due to IL-4, because treatment of macrophages with IL-4 caused down-regulation of PAR1, PAR2, and PAR3. PAR4 mRNA expression remained undetectable in any of the cell types investigated. Stimulation of PAR1, PAR2, and PAR3 with thrombin, trypsin, or established receptor-activating peptides (PAR-APs) triggered cytosolic Ca2+ responses, indicating functionally active PARs. Further, stimulation of monocytes or macrophages with thrombin or PAR1-AP, but not with PAR2-or PAR4-AP, triggers expression of monocyte chemoattractant protein-1 (MCP-1) both at the mRNA and the protein level. These data demonstrate that differentiation of human monocytes is associated with differential expression of functionally active PARs that mediate distinct regulatory functions in inflammation and atherogenesis.     

Human blood monocytes and platelets share a cell surface component

We describe a surface determinant shared by human monocytes and cells of the megakaryocytic axis that has been identified using a mouse monoclonal antibody. This monocyte-platelet antigen (MPA) is expressed on all (greater than 99%) of peripheral blood monocytes, platelets, and megakaryocytes. It is also expressed weakly on the monocytic cell line U937 and the promyelocytic line HL60 and is present on cells from 3 of 4 AML patients examined. It is absent from polymorphonuclear leukocytes, T and B lymphocytes, erythrocytes, and a panel of hematopoietic cell lines. MPA is stripped from monocyte membranes with pronase and is reexpressed overnight. The determinant is carried on a noncovalently linked biomolecular complex with molecular weights of 93,000 and 135,000.     

Alteration in the gene expression pattern of primary monocytes after adhesion to endothelial cells

Monocytes originate from precursors made in the bone and remain in the circulation for nearly 24 h. Much effort has been done to identify the molecules regulating transendothelial migration of monocytes during inflammatory conditions. In contrast, considerably less is known about the process of constitutive monocyte emigration although nearly 340 million monocytes leave the circulation each day in healthy individuals. Previous studies indicated that chemokines were up-regulated in monocytes cocultured with endothelial cells that induce the retraction of the latter cell type, thereby increasing vascular permeability. Thus, we hypothesized that the utilities required for efficient constitutive monocyte extravasation are generated by monocytes themselves because of adhesion to na?ve endothelial cells. To test this hypothesis, cDNA microarray analysis was performed to determine the changes in the gene expression pattern of primary monocytes that have been attached to endothelial cells compared with monocytes that were held in suspension, and we were able to identify three major groups of genes. The first group includes genes such as matrix metalloproteinase 1, monocyte chemoattractant protein 1, and tissue transglutaminase 2, which are likely required for monocyte extravasation. The second group consists of genes that are expressed in phagocytes such as caveolin-1 and CD74. Finally, the third group comprises genes that are expressed in cells of endothelial tissue and cartilage including E-selectin, fibronectin-1, matrix Gla protein, and aggrecanase-2. In summary, we conclude that adhesion of peripheral blood monocytes to na?ve endothelial cells has two effects: mandatory extravasation-specific genes are regulated, and the differentiation program of monocytes is initiated.     

Factor XIII: a marker of mono- and megakaryocytopoiesis

Previous studies have shown that human platelets, megakaryocytes and peripheral blood monocytes contain the subunit a of plasma factor XIII (FXIII) which plays a key role in fibrin stabilization. To study the expression of FXIII subunit a during differentiation of the mono- and megakaryocytic cell lines, bone marrow smears were examined by immunomorphological methods. In addition to megakaryocytes, FXIII was detected in a large number of cells by a highly sensitive immunoperoxidase staining. Characterization of these cells was carried out by double immunofluorescent labelling in which the detection of FXIII subunit a was combined by the labelling of either Leu M3 monocyte/macrophage surface antigen or platelet factor 4 (PF4) a marker for megakaryocytic cells. On the course of differentiation from early precursors to mature megakaryocytes all phenotypic form of the megakaryocytic cell line expressed FXIII subunit a though there were considerable changes in its subcellular distribution pattern. Leu M3 positive cells of monocytopoiesis, i.e. monocytes and promonocytes and in all probability monoblasts as well, were also labelled for FXIII. On this basis FXIII subunit a could be considered as a common marker of megakaryo- and monocytopoiesis and its immunomorphological detection might provide a useful diagnostic tool for identifying normal and perhaps also malignant differentiation forms of these cell lines.     

Heterogeneity of Human Monocytes: An Optimized Four-Color Flow Cytometry Protocol for Analysis of Monocyte Subsets

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5–conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14⁺CD16⁻ monocytes (here termed “Mo1” subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX₃CR1, whereas “nonclassical” CD14^loCD16⁺ monocytes (Mo3) essentially showed the inverse expression pattern. CD14⁺CD16⁺ monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas “nonclassical” monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX₃CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.     

Monocyte activation and T cell inhibition in Plasmodium falciparum-infected placenta

BACKGROUND: During healthy pregnancy, T helper (Th) 1-type and inflammatory-type responses are down-regulated, and Th2-type and proinflammatory-type responses predominate. In Plasmodium falciparum-infected females, these responses induce enhanced production of tumor necrosis factor- alpha and interferon- gamma. METHODS: To assess the respective implication of monocytes and T cells in this placental immunomodulation, we cocultured cells from delivering females living in an area where malaria is endemic. Monocytes and T cells from both peripheral and intervillous blood were crossed in in vitro cultures, to compare the proliferative response to several antigens. Moreover, monocyte cell-surface molecules were quantified by flow cytometry. RESULTS: Coculture results confirmed placental immunomodulation and suggested that the most affected cells are not the intervillous monocytes, which are as able to present the antigen as the peripheral monocytes, but the intervillous T cells. Monocyte staining showed significant increases in human leukocyte antigen D-related, CD54, CD80, and CD86 surface markers in intervillous blood, compared with peripheral blood, which suggests a relative activation of monocytes in the placenta. CONCLUSION: A state of T cell deactivation and monocyte activation is present at delivery. The T cell deactivation in reaction to purified protein derivative could be explained by the presence of local T cell immunoregulatory factors.     

Identification of neutrophil subpopulations with monoclonal antibodies

Two monoclonal antibodies have been produced by the hybridoma technique that recognize subpopulations of human neutrophils. The antibodies, termed 1B5 and 4D1, react with a mean percentage of 57% and 51% of peripheral blood granulocytes, respectively. The antigens recognized appear to be neutrophil specific in that these antibodies do not react with eosinophils, platelets, erythrocytes, monocytes, or nonadherent peripheral blood mononuclear cells. Although the neutrophil subpopulations recognized by these antibodies are nearly identical (coinclusive), the antigenic determinants recognized appear to be different. These monoclonal antibodies to neutrophil subpopulations may prove useful to studying functional heterogeneity among neutrophils as well as for investigations of normal and abnormal myeloid differentiation.     

Acceleration of endothelial-like cell differentiation from CD14+ monocytes in vitro

OBJECTIVE: In vitro differentiation of endothelial cells has potential applications in vascular tissue engineering and cell-based therapy for many diseases. The objective of this study was to develop a new strategy that utilizes cytokines and lipopolysaccharide (LPS) to accelerate endothelial-like cell differentiation from peripheral blood CD14(+) monocytes. METHODS: Peripheral blood CD14(+) monocytes were purified with immunobeads and cultured with an angiogenic growth factor-rich growth medium (EGM-2) with or without initial treatment of LPS in combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 4 days (the day 4 cultures). The cells were then continuously cultured in EGM-2 medium for an additional 4 or 10 days (the day 8 or day 14 cultures). Cell markers were determined by flow cytometry analysis and immunofluorescence staining. Cytokine/chemokine profile was studied by Bio-Plex immunoassay. RESULTS: In the group of initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2, the majority of suspended CD14(+) monocytes were attached and changed their morphology to endothelial-like cells, which expressed high levels of endothelial cell markers CD31, von Willebrand factor, and vascular endothelial growth factor receptor-1 as well as two major endothelial tight junction proteins zonula occludens -1 and occludin in the day 8 cultures. Endothelial nitric oxide synthase expression was substantially increased. Endothelial-like cells were also able to uptake acetylated low-density lipoprotein and bind to Ulex europeus lectin. In addition, endothelial-like cells showed a unique cytokine/chemokine profile with substantial increases of macrophage inflammatory protein-1beta, IL-6, granulocyte colony-stimulating factor, and IL-8. CONCLUSION: Initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2 is an effective strategy for acceleration of endothelial-like cell differentiation from peripheral blood CD14(+) monocytes in vitro.     

Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis

OBJECTIVE: Since it is likely that monocytes utilize chemokines to migrate to the rheumatoid arthritis (RA) joint, we investigated the expression of C-C chemokine receptors (CCR) 1-6 and C-X-C receptor 3 (CXCR3) in the peripheral blood (PB), synovial fluid (SF), and synovial tissue of patients with RA as well as in the PB of normal subjects. METHODS: We compared chemokine receptor expression on CD14+ monocytes from normal PB, RA PB, and RA SF using 2-color flow cytometry. Correlations with patient clinical data were determined. Chemokine and receptor expression were investigated in RA synovial tissue by immunohistochemistry and 2-color immunofluorescence to identify CD68+ macrophages. RESULTS: Most normal PB monocytes expressed CCR1 (87%) and CCR2 (84%), but not CCRs 3, 4, 5, or 6 or CXCR3. RA PB monocytes expressed CCR1 (56%) and CCR2 (76%), with significantly more expressing CCR3 (18%), CCR4 (38%), and CCR5 (17%) compared with normal PB monocytes. Significantly fewer SF monocytes from RA patients expressed CCR1 (17%), CCR2 (24%), and CCR4 (6%) while significantly more expressed CCR3 (35%) and CCR5 (47%) compared with RA and normal PB monocytes; CCR6 and CXCR3 were rarely detected. Clinically, the erythrocyte sedimentation rate was inversely correlated with the expression of CCR1 and CCR4 by RA PB, and CCR5 expression by RA SF was correlated with the SF white blood cell count. CCR1-, CCR2-, and CCR5-immunoreactive cells were found in RA synovial tissue and colocalized with CD68+ macrophages. RA synovial tissue RANTES (regulated upon activation, normally T cell expressed and secreted chemokine)- and monocyte chemoattractant protein 1-immunoreactive cells colocalized with CCR1 and CCR2, respectively, on serial sections. Macrophage inflammatory protein 1alpha (MIP-1alpha) was principally restricted to vascular endothelium, and MIP-1beta+ macrophages were found throughout the sections. CONCLUSION: Monocytes mainly express CCR1 and CCR2 in normal and RA PB, CCR3 and CCR5 in RA PB and RA SF, and CCR4 in RA PB. The differential expression of chemokine receptors suggests that certain receptors aid in monocyte recruitment from the circulation while others are important in monocyte retention in the joint.     

Monocytes circulate in constant reversible interaction with platelets in a [Ca2+]-dependent manner

Abstract

We aimed to provide evidence that blood monocytes belonging to all subsets predominantly circulate in constant and usually reversible interactions with platelets, which are predominantly [Ca2+] dependent. The proportions of monocyte–platelet aggregates (MPAs) attributable to individual monocyte subsets in fresh and promptly processed heparin-anticoagulated blood from 10 healthy subjects (median age 35 years, 50% male) were analysed by flow cytometry and compared to samples anticoagulated with a potent [Ca2+] chelator, ethylenediaminetetraacetic acid (EDTA). Additional experiments with [Ca2+] depletion or supplementation were also performed. Monocytes subsets were defined as CD14++CD16–CCR2+ cells (Mon1), CD14++CD16+CCR2+ cells (Mon2) and CD14+CD16++CCR2? cells (Mon3). Vast majority of monocytes showed aggregation with platelets in heparinised samples, but most monocytes were free of platelets when EDTA was used (p?p?=?0.005 for all subsets). Supplementation with CaCl₂ resulted in dose-dependent increase in MPAs (p?p?p?=?0.004). In healthy subjects monocytes circulate in constant, but predominantly reversible and [Ca2+]-dependent aggregation with platelets. These observations may reflect a complex involvement of platelets in regulation of monocyte activity.