Circulating CD2+ monocytes are dendritic cells

Low levels of CD2 have been described on subsets of monocytes, macrophages, and dendritic cells. CD2 is expressed on about one-third of circulating monocytes, at levels one-half log lower than on T or NK cells, representing 2-4% of PBMC. FACS analysis of CD2+ and CD2- monocytes revealed no significant difference in the expression of adhesion molecules (CD11a/b/c), class II Ags (HLA-DR, -DQ, -DP), myeloid Ags (CD13, CD14, CD33), or costimulatory molecules (CD80, CD86). Freshly isolated CD2+ and CD2- monocytes were morphologically indistinguishable by phase contrast microscopy. However, scanning electron microscopy revealed large prominent ruffles on CD2+ monocytes in contrast to small knob-like projections on CD2- monocytes. After 2 days of culture, the CD2+ monocytes largely lost CD14 expression and developed distinct dendrites, whereas the CD2- monocytes retained surface CD14 and remained round or oval. Freshly isolated CD2+ monocytes were more potent inducers of the allogeneic MLR and more efficiently induced proliferation of naive T cells in the presence of HIV-1 gp120 than did CD2- monocytes. After culture in the presence of GM/CSF and IL-4, CD2+ monocytes were up to 40-fold more potent than monocyte-derived dendritic cells or CD2- monocytes at inducing allogeneic T cell proliferation. These findings suggest that circulating CD2+ and CD2- monocytes are dendritic cells and the precursors of macrophages, respectively. Thus, dendritic cells are far more abundant in the blood than previously thought, and they and precursors of macrophages exist in the circulation as phenotypically, morphologically, and functionally distinct monocyte populations.     

Beryllium Increases the CD14dimCD16+ Subset in the Lung of Chronic Beryllium Disease

CD14^dimCD16+ and CD14^brightCD16+ cells, which compose a minor population of monocytes in human peripheral blood mononuclear cells (PBMC), have been implicated in several inflammatory diseases. The aim of this study was to investigate whether this phenotype was present as a subset of lung infiltrative alveolar macrophages (AMs) in the granulomatous lung disease, chronic beryllium disease (CBD). The monocytes subsets was determined from PBMC cells and bronchoalveolar lavage (BAL) cells from CBD, beryllium sensitized Non-smoker (BeS-NS) and healthy subjects (HS) using flow cytometry. The impact of smoking on the AMs cell phenotype was determined by using BAL cells from BeS smokers (BeS-S). In comparison with the other monocyte subpopulations, CD14^dimCD16+ cells were at decreased frequency in PBMCs of both BeS-NS and CBD and showed higher HLA-DR expression, compared to HS. The AMs from CBD and BeS-NS demonstrated a CD14^dimCD16+phenotype, while CD14^brightCD16+ cells were found at increased frequency in AMs of BeS, compared to HS. Fresh AMs from BeS-NS and CBD demonstrated significantly greater CD16, CD40, CD86 and HLA-DR than HS and BeS-S. The expression of CD16 on AMs from both CBD and BeS-NS was downregulated significantly after 10μM BeSO₄ stimulation. The phagocytic activity of AMs decreased after 10μM BeSO₄ treatment in both BeS-NS and CBD, although was altered or reduced in HS and BeS-S. These results suggest that Be increases the CD14^dimCD16+ subsets in the lung of CBD subjects. We speculate that Be-stimulates the compartmentalization of a more mature CD16+ macrophage phenotype and that in turn these macrophages are a source of Th1 cytokines and chemokines that perpetuate the Be immune response in CBD. The protective effect of cigarette smoking in BeS-S may be due to the low expression of co-stimulatory markers on AMs from smokers as well as the decreased phagocytic function.     

Characterization of the CD14++CD16+ Monocyte Population in Human Bone Marrow

Numerous studies have divided blood monocytes according to their expression of the surface markers CD14 and CD16 into following subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and nonclassical CD14⁺CD16⁺⁺ monocytes. These subsets differ in phenotype and function and are further correlated to cardiovascular disease, inflammation and cancer. However, the CD14/CD16 nature of resident monocytes in human bone marrow remains largely unknown. In the present study, we identified a major population of CD14⁺⁺CD16⁺ monocytes by using cryopreserved bone marrow mononuclear cells from healthy donors. These cells express essential monocyte-related antigens and chemokine receptors such as CD11a, CD18, CD44, HLA-DR, Ccr2, Ccr5, Cx3cr1, Cxcr2 and Cxcr4. Notably, the expression of Ccr2 was inducible during culture. Furthermore, sorted CD14⁺⁺CD16⁺ bone marrow cells show typical macrophage morphology, phagocytic activity, angiogenic features and generation of intracellular oxygen species. Side-by-side comparison of the chemokine receptor profile with unpaired blood samples also demonstrated that these rather premature medullar monocytes mainly match the phenotype of intermediate and partially of (non)classical monocytes. Together, human monocytes obviously acquire their definitive CD14/CD16 signature in the bloodstream and the medullar monocytes probably transform into CD14⁺⁺CD16⁻ and CD14⁺CD16⁺⁺ subsets which appear enriched in the periphery.     

外周血CD14~(high)CD16~+单核细胞在HIV/HCV重叠感染者肝损伤中的作用

目的:探讨HIV/HCV重叠感染患者外周血单核细胞亚群与肝损伤的关系。方法:观察对象为HIV/HCV重叠感染患者,分为对照组(n=11)、肝纤维化组(n=12)和肝硬化组(n=7)。运用流式细胞仪检测单核细胞及其亚群变化,瞬时弹性成像(Fibroscan)检测肝纤维化情况。比较单核细胞各亚群在不同程度肝损伤中的差异,并对HIV/HCV重叠感染患者外周血的单核细胞数与肝纤维化情况进行相关性分析。结果:HIV/HCV重叠感染患者肝硬化组与对照组比较,单核细胞CD14low CD16+和CD14high CD16+亚群显著增多(P=0.047,P=0.018)。HIV/HCV重叠感染患者肝纤维化组与对照组比较,单核细胞各亚群差异无统计学意义(P=0.84,P=0.812)。HIV/HCV重叠感染患者CD14high CD16+单核细胞与肝纤维化情况存在正性线性相关,方程成立,并且系数有统计学意义(P=0.018),方程似然比(r 2)0.45。结论:HIV/HCV重叠感染患者CD14high CD16+单核细胞增高有可能是肝损伤加重的原因之一。     

A CD1a+/CD11c+ subset of human blood dendritic cells is a direct precursor of Langerhans cells.

Based on the relative expression of CD11c and CD1a, we have identified three fractions of dendritic cells (DCs) in human peripheral blood, including a direct precursor of Langerhans cells (LCs). The first two fractions were CD11c+ DCs, comprised of a major CD1a+/CD11c+ population (fraction 1), and a minor CD1a-/CD11c+ component (fraction 2). Both CD11c+ fractions displayed a monocyte-like morphology, endocytosed FITC-dextran, expressed CD45RO and myeloid markers such as CD13 and CD33, and possessed the receptor for GM-CSF. The third fraction was comprised of CD1a-/CD11c- DCs (fraction 3) and resembled plasmacytoid T cells. These did not uptake FITC-dextran, were negative for myeloid markers (CD13/CD33), and expressed CD45RA and a high level of IL-3Ralpha, but not GM-CSF receptors. After culture with IL-3, fraction 3 acquired the characteristics of mature DCs; however, the expression of CD62L (lymph node-homing molecules) remained unchanged, indicating that fraction 3 can be a precursor pool for previously described plasmacytoid T cells in lymphoid organs. Strikingly, the CD1a+/CD11c+ DCs (fraction 1) quickly acquired LC characteristics when cultured in the presence of GM-CSF + IL-4 + TGF-beta1. Thus, E-cadherin, Langerin, and Lag Ag were expressed within 1 day of culture, and typical Birbeck granules were observed. In contrast, neither CD1a-/CD11c+ (fraction 2) nor CD1a-/CD11c- (fraction 3) cells had the capacity to differentiate into LCs. Furthermore, CD14+ monocytes only expressed E-cadherin, but lacked the other LC markers after culture in these cytokines. Therefore, CD1a+/CD11c+ DCs are the direct precursors of LCs in peripheral blood.     

CD4+CD45RA+ and CD4+CD45RA- T cell subsets in man maintain distinct function and CD45RA expression persists on a subpopulation of CD45RA+ cells after activation with Con A.

We have previously shown that Con A-induced suppressor T cells belong to the CD45RA+ subset. After unseparated T cells are activated with Con A, CD45RA expression increases to a maximum (Day 2), and then decreases significantly, but does not disappear entirely (Day 9), while CD29 expression increases steadily. In the present study, we examined the fate of these cell surface molecules on isolated CD4+CD45RA+ and CD4+CD45RA- cells following activation with Con A, and their relationship to the regulatory functions of these subsets. After activation of CD4+CD45RA+ cells with Con A, CD45RO and CD29 antigen expression rapidly increases (greater than 90%). While CD45RA expression is downregulated, approximately 40% of the cells continue to express low-density CD45RA in a stable fashion through Day 21. Despite these phenotypic changes, cells originally CD45RA+ continue to suppress IgG synthesis and provide only minimal B cell help. Furthermore, when cells originally CD45RA+ were sorted on the basis of continued presence, or loss of CD45RA antigen 14 days after activation, both populations demonstrated potent suppression and minimal help. In contrast, after activation with Con A, CD4+CD45A- cells maintain stable phenotype and provide significant help and minimal suppression. Immunoprecipitation of the CD45RA antigen from Day 14 activated CD4+CD45RA+ cells confirms the continued presence of the 205-kDa isoform, but reveals a significant decrease in the 220-kDa isoform. These results suggest that after activation with Con A, cells originally CD45RA+ remain functionally distinct from cells originally CD45RA-, and that CD45RA antigen persists on a subpopulation of CD45RA+ cells after activation with Con A.     

TCRαβ^＋CD^＋4CD^—8胸腺髓质细胞的表型分析

Phenotypic analysis of the medullary-type CD4+CD8- (CD4SP) thymocytes have revealed phenotypic heterogeneity within these cells. The phenotype of mature peripheral T cells is Qa-2+ HSA- CD69-, whereas in the medullary-type CD4SP thymocytes, the expression pattern of many markers were quite different, suggesting that the medullary-type CD4SP thymocytes may undergo phenotypic maturation. According to the results of two-color cytometry, seven discrete phenotypes were defined by the relative expression of Qa-2, HSA, CD69, 3G11 and 6C10: 3G11-6C10+CD69+HSAhi-->3G11+6C10+CD69+ HSAhi-->3G11+6C10-CD69+HSAint-->3G11+6C10- CD69-HSAint Qa-2(-)-->3G11+HSAlo/-Qa-2lo, at the same time, 3G11+6C10-CD69-HSAint Qa-2(-)-->3G11-HSAlo Qa-2(-)-->3G11-HSAlo/- Qa-2hi, the last two Qa-2 positive subsets could exit the thymus and home into periphery.     

Senescent CD14+CD16+ monocytes exhibit proinflammatory and proatherosclerotic activity

In elderly subjects and in patients with chronic inflammatory diseases, there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype, whose origin and functional relevance has not been well characterized. In this study, we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells, and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence, intracellular cytokine expression, ability to interact with endothelial cells, and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p < 0.05) and increased expression of β-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%, respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209, release of cytokines in response to low-intensity stimulus, and increased capacity to sustain lymphocyte proliferation. Finally, compared with CD14(++)CD16(-) cells, CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary, our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells, with increased inflammatory activity and ability to interact with endothelial cells. Therefore, accumulation of senescent monocytes may explain, in part, the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases.     

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor G?6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.     

Innate immune responses to herpes simplex virus type 2 influence skin homing molecule expression by memory CD4+ lymphocytes

Herpes simplex virus (HSV) infections of humans are characterized by intermittent, lytic replication in epithelia. Circulating HSV-specific CD4 T cells express lower levels of preformed cutaneous lymphocyte-associated antigen (CLA), a skin-homing receptor, than do circulating HSV-specific CD8 T cells but, paradoxically, move into infected skin earlier than CD8 cells. Memory CD4 T cells develop strong and selective expression of CLA and E-selectin ligand while responding to HSV antigen in vitro. We now show that interleukin-12, type I interferon, and transforming growth factor beta are each involved in CLA expression by memory HSV type 2 (HSV-2)-specific CD4 T cells in peripheral blood mononuclear cells (PBMC). A reduction of the number of monocytes and dendritic cells from PBMC reduces CLA expression by HSV-2-responsive CD4 lymphoblasts, while their reintroduction restores this phenotype, identifying these cells as possible sources of CLA-promoting cytokines. Plasmacytoid dendritic cells are particularly potent inducers of CLA on HSV-reactive CD4 T cells. These observations are consistent with cooperation between innate and acquired immunity to promote a pattern of homing receptor expression that is physiologically appropriate for trafficking to infected tissues.     

Increased susceptibility to apoptosis of CD56dimCD16+ NK cells induces the enrichment of IFN-gamma-producing CD56bright cells in tuberculous pleurisy

Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16- subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-gamma production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.     

Monocyte-induced down-modulation of CD16 and CD56 antigens on human natural killer cells and its regulation by histamine H2-receptors.

Human natural killer (NK) cells carry CD16/FcR and CD56 cell-surface Ag but lack the T-cell marker CD3. Here we show that incubation of resting human NK cells with CD3-/16+/56+ phenotype with autologous monocytes induced the disappearance of CD16 and CD56 cell-surface Ag on NK-cells but did not affect CD2 or CD3 Ag expression on T-cells. Monocyte-induced down-modulation of NK-cell-surface Ag was cell-contact dependent and induced only by freshly isolated monocytes, recovered from peripheral blood by counter-current centrifugal elutriation. Adherence of monocytes abrogated the capacity to induce down-modulation of NK-cell-surface Ag. The biogenic amine histamine dose-dependently reversed the monocyte-induced down-modulation of CD16 and CD56 on CD3- NK-cells. The effect of histamine was mediated by H2-type receptors on monocytes. The data presented are suggestive of a cell-cell-mediated interaction between monocytes and NK-cells which modulates surface expression of NK-cell Ag and its histaminergic regulation.     

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.     

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells

BACKGROUND: Transplantable B16 melanoma is widely used as a tumor model to investigate tumor immunity. We wished to characterize the leukocyte populations infiltrating B16 melanoma tumors, and the functional properties of tumor-infiltrating dendritic cells (TIDC). MATERIALS AND METHODS: We used the B16 melanoma cell line expressing ovalbumin protein (OVA) to investigate the phenotype and T cell stimulatory capacity of TIDC. RESULTS: The majority of leukocytes in B16 melanoma were macrophages, which colocalized with TIDCs, B and T cells to the peripheral area of the tumor. Both myeloid and plasmacytoid DC populations were present within tumors. Most of these DCs appeared immature, but about a third expressed a mature phenotype. TIDCs did not present tumor-derived antigen, as they were unable to induce the proliferation of tumor-specific CD4+ and CD8+ T cells in vitro unless in the presence of specific peptides. Some presentation of tumor-derived antigen could be demonstrated in the tumor-draining lymph node using in vivo proliferation assays. However, while proliferation of CD8+ T cells was reproducibly demonstrated, no proliferation of CD4+ T cells was observed. CONCLUSION: In summary, our data suggest that DCs in tumors have limited antigen-presenting function. Inefficient antigen presentation extends to the tumor-draining lymph node, and may affect the generation of antitumor immune responses.     

CXCR1+CD4+ T cells in human allergic disease

Chemokine receptors play an important role in the migration of leukocytes to sites of allergic inflammation in humans. In this study, we have identified increased expression of the chemokine receptor CXCR1 on CD4+ T lymphocytes derived from patients with atopic disease compared with normal donors. Enhanced expression of CXCR1 by atopic donors was identified on freshly isolated peripheral blood cells and on expanded cell populations derived from nasal mucosal biopsies and from the periphery. Identification of CXCR1 expression on CD4 cells in the nasal mucosa was confirmed by double immunofluorescence. In addition, expression of CXCR1 was dramatically decreased in patients undergoing successful treatment of allergic rhinitis by specific immunotherapy. CXCR1 provided a functional receptor capable of regulating T cells in the context of allergic disease, since expression of CXC chemokine ligand 8 was up-regulated at the site of allergic inflammation and freshly isolated CXCR1+CD4+ cells from atopic donors showed an enhanced functional response to this ligand. CXCR1 expression on CD4+ T cells was increased in vitro in response to the pro-Th2 cytokine IL-4. Phenotypic analysis reveals that IFN-gamma expression was lower in the CXCR1+CD4+ cells. The identification of CXCR1 as a marker of allergic rhinitis reveals a possible target for therapeutic intervention in atopic disease.     

The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF

In human blood two monocyte populations can be distinguished, i.e., the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14(+)CD16(+) monocytes are major producers of TNF in human blood.     

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.     

Soluble form of the endothelial adhesion molecule CD146 binds preferentially CD16+ monocytes

The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16−, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62–3.87) and of CD14+/CD16+ (2.59, 1.28–4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16− (0.66, 0.47–1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16− monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.     

Differentiation to the CCR2+ inflammatory phenotype in vivo is a constitutive, time-limited property of blood monocytes and is independent of local inflammatory mediators

It is proposed that CCR2+ monocytes are specifically recruited to inflammatory sites, whereas CCR2- monocytes are recruited to normal tissue to become resident macrophages. Whether these subsets represent separate lineages, how differential trafficking is regulated and whether monocytes undergo further differentiation is uncertain. Using a mouse model of autoimmune uveoretinitis we examined monocyte trafficking to the inflamed retina in vivo. We show that bone marrow-derived CD11b+ F4/80- monocytes require 24 to 48 h within the circulation and lymphoid system before acquiring the CCR2+ phenotype and trafficking to the inflamed retina is enabled. This phenotype, and the capacity to traffic were lost by 72 h. Monocyte CCR2 expression followed a similar time course in normal mice indicating that differentiation to an inflammatory phenotype is a constitutive, time-limited property, independent of local inflammatory mediators. Phenotypic analysis of adoptively transferred cells indicated that circulating inflammatory monocytes also differentiate into CD11c+ and B220+ dendritic cells and F4/80+ tissue macrophages in vivo. Our data supports the hypothesis of continuous extravasation and progressive differentiation over time of inflammatory monocytes in the circulation rather than replication within the actively inflamed tissue, and supports the concept of myeloid dendritic cell differentiation from trafficking monocytes under physiological conditions in vivo.