Radiation damage to endothelial cells in vitro, as judged by the micronucleus assay

CD23 has been reported to be a macrophage/monocyte activation antigen. We focused on the expression of CD23 by peripheral blood macrophages/monocytes in 5 Kawasaki disease (KD) patients with coronary artery lesions (CAL) and compared these values with those of 35 patients without CAL. The expression of CD23 on peripheral blood macrophages/monocytes was assayed by a fluorescence-activated cell sorter using monoclonal antibodies CD23 and CD14. Absolute counts of CD23+CD14+ macrophages/monocytes in KD patients with CAL did not increase during the acute stage, while these values in KD patients without CAL increased. In addition, this decreased expression of CD23 on peripheral blood macrophages/monocytes in patients with CAL did not change during the acute stage, regardless of IVGG therapy. Our results suggest that the decreased expression of CD23 on peripheral blood macrophages/monocytes in patients with CAL is part of the regulatory system of CD23 antigen during acute KD.     

Efficacy of traditional Chinese herbal therapy in vitro. A model system for atopic eczema: inhibition of CD23 expression on blood monocytes

Recently, there has been growing interest in the use of traditional Chinese herbal therapy (TCHT) decoctions for the treatment of atopic eczema (AE). The mode of action of this treatment is still unknown, and in order to investigate this we have analysed the effect of an extract of these herbs (TCHTE) on interleukin 4 (IL-4)-induced CD23 expression on peripheral blood monocytes from non-atopic subjects. We found that TCHTE inhibited CD23 expression up to 60% (P < 0.001), whereas the placebo extract had no significant effect on CD23 expression. This inhibition was dose-dependent, and TCHTE was effective at a concentration of 250 micrograms/ml (P = 0.001). If TCHTE or placebo was added after IL-4, the action of TCHTE could still be seen at 12 h. This inhibition was not due to cell death, as peripheral blood mononuclear cells (PBMCs) cultured with TCHTE or placebo at a concentration used in these experiments had a similar viability to control cultures. Down-regulation of the low affinity receptors for IgE on antigen-presenting cells in patients with AE may contribute to the benefit observed following treatment with TCHT.     

Reducing financial barriers to HIV-related medical care: does the Ryan White CARE Act make a difference?

OBJECTIVE: To assess the CD23 status in patients with juvenile chronic arthritis (JCA), as defined by serum soluble CD23 (sCD23) and the expression of CD23 on peripheral blood mononuclear cells. METHODS: Serum sCD23 levels were measured by ELISA in 22 patients with systemic JCA (s-JCA), in 40 patients with antinuclear antibody positive pauciarticular JCA (ANA+ p-JCA), and in 38 healthy controls. CD23 expression on T cells, B cells, and monocytes was determined by flow cytometry analysis of double fluorescence staining. RESULTS: Serum sCD23 levels were increased in both ANA+ p-JCA and s-JCA; no relation with disease activity or severity was found. In patients with ANA+ p-JCA, serum sCD23 levels were correlated with an increased percentage of B cells expressing CD23, while in patients with s-JCA the serum sCD23 levels were correlated with an increased percentage of monocytes expressing CD23. CONCLUSION: Serum sCD23 levels are elevated both in systemic and ANA+ pauciarticular JCA: different cell subset CD23 expression in s-JCA and ANA+ p-JCA (i.e. monocyte or B cell, respectively) suggests that in pauciarticular JCA CD23 may be implicated in B cell activation and autoantibody production, while in systemic JCA may be involved in monocyte activation and in the release of inflammatory mediators.     

Cytokine-regulated expression of activated leukocyte cell adhesion molecule (CD166) on monocyte-lineage cells and in rheumatoid arthritis synovium

OBJECTIVE: To determine whether monocyte/macrophage expression of the CD6 ligand, activated leukocyte cell adhesion molecule (ALCAM) (CD166), is regulated by cytokines during inflammation in rheumatoid arthritis (RA). METHODS: We used flow cytometry to test whether cytokines present in rheumatoid synovium could regulate ALCAM cell surface expression on peripheral blood (PB) monocytes and RA synovial fluid (SF) macrophages, and we examined ALCAM expression in situ in RA synovium by immunofluorescence. RESULTS: The monocyte differentiation factors interleukin-3, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor augmented ALCAM expression on PB monocytes. ALCAM was expressed on monocyte-lineage cells in situ in inflamed synovium from patients with RA (9 of 9), but not in uninflamed synovium from patients with joint trauma (0 of 3). Furthermore, in vitro culture-induced ALCAM expression on PB monocytes and CD14+ RA SF cells was inhibited by an M-CSF neutralizing antibody. CONCLUSION: ALCAM expression on PB and SF monocytes/macrophages is enhanced by M-CSF.     

Measuring temperature and humidity in the breathing circuit

Upregulation of adhesion molecule expression on endothelial cells (EC) and circulating leukocytes, by locally produced inflammatory mediators, may result in the enhanced infiltration of leukocytes into tissue, e.g. the airways of asthma patients. The present study investigates whether the expression of adhesion molecules on granulocytes and monocytes from asthma patients is affected by chemotactic factors, i.e. interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1). Flow cytometric analysis showed that the intrinsic expression of the various adhesion molecules on peripheral blood phagocytes from asthma patients was not different from that of healthy individuals. However, stimulation of monocytes with MCP-1 resulted only in upregulation of the expression of CD14 on monocytes from symptomatic asthma patients but not on monocytes from asymptomatic asthma patients and healthy individuals. Stimulation of granulocytes with IL-8 did not change the expression of the various beta 1- and beta 2-integrin molecules, such as VLA-4, LFA-1, CR3 and p150,95. Since earlier studies have shown that CD14 on monocytes mediates monocyte adhesion to activated vascular EC the present findings suggest that during the active phase of asthma upregulation of CD14 on monocytes by MCP-1 may lead to an increased adhesion of monocytes to vascular endothelium and their subsequent transendothelial migration into the tissue of the airways.     

Molecular analysis of the adaptive response of intestinal bile acid transport after ileal resection in the rat

OBJECTIVES: Major operative trauma like aorta-coronary bypass operation may lead to postoperative immunodisturbance, putting the patient at an increased risk for infection and sepsis. The monocyte/macrophage system and the endotoxin receptor CD14 are important in the early recognition and elimination of invading bacteria. The aim of this study was to analyze changes in membrane-associated CD14 and soluble CD14 during and after cardiac involving cardiopulmonary bypass. METHODS: We studied numbers of leukocytes, monocytes, and monocyte subpopulations, expression of monocyte membrane-associated CD14 and plasma levels of soluble CD14 in 10 patients (63 +/- 8 years of age), who underwent elective cardiopulmonary bypass. RESULTS: Cardiopulmonary bypass induced marked postoperative monocytosis, which was maximal 20 hours after the operation (485 +/- 242 cells/microl before, 1080 +/- 264 cells/microl 20 hours after surgery). Expression of membrane-associated CD14 on classical CD14++ monocytes decreased significantly by 40%, reaching a nadir 20 hours after surgery (p < 0.05). At the time of maximal membrane-associated CD14 suppression, the levels of soluble CD14 measured by enzyme-linked immunosorbent assay were clearly increased (3.2 +/- 1.0 microg/ml before versus 5.6 +/- 1.0 microg/ml 20 hours after, p < 0.001). No significant change of the percentage of small (alpha) and large (beta) forms of soluble CD14 was found. CONCLUSIONS: Cardiopulmonary bypass leads to reduced membrane-associated CD14 expression on peripheral blood monocytes and increased levels of soluble CD14 through shedding or secretion of membrane-associated CD14 from the cell surface. These findings indicate that bypass is associated with significant monocyte activation.     

In vivo relevance of CD30 in atopic dermatitis

CD30 expression was evaluated by immunohistochemistry in lesional skin biopsies of eight patients with active atopic dermatitis (AD) and three patients with allergic contact (nickel-induced) dermatitis (ACD). CD30 expression was also assessed in a large panel of CD4+ and CD8+ T-cell clones generated from the skin biopsies of four patients with AD. Finally, the levels of soluble CD30 (sCD30) were measured in the serum of 41 patients with AD, 19 patients with ACD, and 60 healthy controls. In all specimens of lesional AD skin, where the great majority of infiltrating cells were CD4+ T cells, remarkable numbers of cells were CD30+, whereas virtually no CD30+ cells were found in the skin of patients with ACD. In CD4+ T-cell clones generated from the lesional AD skin, most of which produced both interleukin (IL)-4 and interferon-gamma (IFN-gamma) (Th0-like cells) or IL-4 and IL-5, but not IFN-gamma (Th2-like cells), CD30 expression directly correlated with the ability to produce IL-4 and IL-5, but was inversely related to IFN-gamma production. High levels of sCD30 (correlated with disease activity: r = 0.618) were detected in the serum of most AD patients, whereas there was no increase of sCD30 levels in the serum of patients with ACD. These data support the view that Th0/Th2-type responses predominate in the skin of patients with AD and suggest that the presence of CD30+ T cells in tissues and/or increased levels of sCD30 in biologic fluids are indicative of Th2-dominated responses.     

Increased expression of costimulatory molecules on peripheral blood monocytes in patients with Crohn's disease

BACKGROUND: Activation of T lymphocytes and monocytes/macrophages has been implicated in the pathogenesis of Crohn's disease (CD). Costimulatory molecules play important roles in optimal T-cell activation. METHODS: With flow cytometric analysis we have investigated the expression of the costimulatory molecules B7-1 (CD80), B7-2 (CD86), and CD18 and the intercellular adhesion molecule-1 (ICAM-1) on peripheral blood monocytes and the expression of the activation markers HLA-DR and IL-2R (CD25) on peripheral blood T lymphocytes from 31 CD patients, 17 ulcerative colitis (UC) patients, and 10 healthy controls. RESULTS: In CD patients the percentage of activated T cells (CD3+ HLA-DR+ and CD3+ IL-2R+) was significantly increased compared with those of controls and UC patients (P < 0.05). Most monocytes from all three groups expressed B7-2, CD18, and ICAM-1 molecules (all > 79%), but only a few positive cells expressed B7-1 molecules (< 5%). No significant differences were detected in the percentage positivity of all costimulatory molecules tested among CD, UC, and controls. The mean fluorescence intensity (MFI) of B7-1 in all three groups was very weak and not significantly different. However, in CD patients there was a significantly increased MFI of B7-2, CD18, and ICAM-1 molecules compared with UC and controls (P < 0.05). On the other hand, both the percentage positivity and MFI of HLA-DR molecules on monocytes of UC patients were significantly lower than those of CD patients and controls (P < 0.05). CONCLUSIONS: Expression of the costimulatory molecules B7-2, CD18, and ICAM-1 on peripheral blood monocytes of CD patients is increased. In CD patients activation of peripheral T lymphocytes may correlate with increased expression of these costimulatory molecules on peripheral blood monocytes.     

Expression and release of the monocyte lipopolysaccharide receptor antigen CD14 are suppressed by glucocorticoids in vivo and in vitro

The effect of glucocorticoid (GC) treatment on expression and release of the monocyte cell surface LPS receptor Ag CD14 was studied in vivo and in vitro. In patients with acute inflammatory diseases receiving GC pulse therapy serum concentrations of soluble CD14 and CD14 expression by peripheral blood monocytes decreased significantly. The LPS-binding capacity correlated positively with the amount of cell surface CD14 by human blood monocytes. In vitro, a time- and dose-dependent effect of GC preparations on monocyte membrane and soluble CD14 by cultured peripheral blood monocytes was found. Incubation with 2 x 10(-8) M prednisolone down-regulated cell surface CD14 after 72 h, and 2 x 10(-7) M suppressed CD14 expression even after 24 h. Prednisolone also decreased release of the soluble CD14 Ag, where a 10-fold higher GC concentration was required for a significant suppression compared with membrane CD14 during culture. Expression of other monocyte membrane Ags were either unchanged (CD33, CD35), diminished (CD13, CD89), or increased (CD32) by GC, indicating no general down-modulation of cell surface Ag expression. Preincubation with glucocorticoids for 24 h significantly down-regulated CD14 expression during subsequent steroid-free culture for at least 7 days. In cultured monocytes, the LPS-induced increase of membrane and soluble CD14 was markedly but not completely inhibited by prednisolone. Therefore, GC treatment suppresses the up-regulation of the LPS receptor during endotoxin challenge, and likewise, the IL-1 secretion after LPS stimulus was significantly diminished. Taken together, the suppression of the monocytic cell surface and soluble endotoxin receptor CD14 by GC may contribute to the increased risk of infections in patients undergoing steroid therapy.     

Leukocyte phenotypic changes in an in vitro model of ABO hemolytic transfusion reaction

BACKGROUND: ABO antigen-antibody interaction in the presence of peripheral blood leukocytes (white cells) results in the production of a variety of proinflammatory cytokines. However, although tumor necrosis factor alpha has been shown to be derived at least primarily from monocytes, the range of cells activated by this process has not previously been reported. Therefore, changes in mononuclear cell surface antigen expression were studied, to determine which subsets of white cells appeared to be activated in the setting of ABO incompatibility. STUDY DESIGN AND METHODS: Group O peripheral blood mononuclear cells (PBMCs) were incubated in autologous plasma with group A or O red cells (RBCs) for up to 24 hours. White cell expression of activation and adhesion markers was measured at 2 and 24 hours by flow cytometry, using direct or indirect fluorescein or phycoerythrin labeling. RESULTS: Expression of lymphocyte activation markers CD25, CDw108, and CD109 was equivalent when PBMCs incubated with group A and O RBCs were compared. However, after 2 hours, mean fluorescence of CD14 on PBMCs incubated with group A RBCs was 65 percent of that on PBMCs incubated with group O RBCs and remained similarly decreased at 24 hours. CD44 expression was upregulated on PBMCs exposed to both group A and O RBCs, but it was increased significantly more on monocytes exposed to group A RBCs. The ability to bind hyaluronic acid was induced in approximately 42 percent of CD14+ monocytes exposed to group A RBCs but in no cells exposed to group O RBCs. CONCLUSION: Downregulation of CD14 and increased binding of hyaluronic acid reflects monocyte activation in this model. No evidence of lymphocyte activation was found, supporting the hypothesis that ABO transfusion reactions primarily activate monocytes.     

Unique monocyte subset in patients with AIDS dementia

BACKGROUND: 15-30% of patients infected with HIV will develop a debilitating dementia. Whilst HIV enters the brain soon after infection, presumably within monocyte-derived macrophages, not all patients with HIV become demented. Blood monocytes probably cross the blood-brain barrier and give rise ultimately to parenchyma macrophages. We looked for a specific monocyte subset in AIDS patients with dementia. METHODS: Peripheral blood monocytes from three groups were compared: AIDS patients with (n = 12) and without (n = 11) dementia, and ten HIV seronegative healthy controls. We used flow cytometry to analyse monocytes, and cell lysis and apoptosis assays to examine monocyte effects on human brain cells in vitro. FINDINGS: We found a unique subset of monocytes in patients with AIDS dementia. These monocytes were more dense and granular and expressed CD14/CD16 and CD14/CD69. Means (SD) for CD14/CD16 in HIV-negative controls and in AIDS non-dementia and AIDS dementia patients were 6.5% (4), 16% (13), and 37% (21), respectively (p = 0.008 between the two groups of patients). The corresponding means for CD14/CD69 were 7% (6), 8% (10), and 69% (18) (p < 0.0001). INTERPRETATION: CD69 is a member of the natural-killer-cell gene complex that is expressed after activation. Supernatants from cultures containing these dense cells can trigger apoptosis of human brain cells in vitro. The monocyte subset we found in patients with AIDS dementia might enter the brain and expose neural cells to toxic factors.     

Monocytes from Wiskott-Aldrich patients display reduced chemotaxis and lack of cell polarization in response to monocyte chemoattractant protein-1 and formyl-methionyl-leucyl-phenylalanine

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by trombocytopenia, eczema, and progressive decline of the immune function. In addition, lymphocytes and platelets from WAS patients have morphologic abnormalities. Since chemokines may induce morphologic changes and migration of leukocytes, we investigated the monocyte response to chemoattractants in cells from WAS patients with an identified mutation in the WAS protein gene. Here, we report that monocytes derived from four patients with molecularly defined typical WAS have a severely impaired migration in response to FMLP and to the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha compared with normal donors. Conversely, neither MCP-1 binding to monocytes nor induction of the respiratory burst by MCP-1 and FMLP is significantly different between WAS patients and normal donors. Within a few minutes of stimulation, monocytes respond to chemokines with increased expression of adhesion molecules and with morphologic changes such as cell polarization. Although up-regulation of CD11b/CD18 expression following stimulation with FMLP or MCP-1 is preserved in WAS patients, cell polarization is dramatically decreased. Staining of F-actin by FITC-phalloidin in monocytes stimulated with chemoattractants shows F-actin to have a rounded shape in WAS patients, as opposed to the polymorphic distribution of F-actin in the polarized monocytes from healthy donors. These results suggest that WAS protein is involved in the monocyte response to the chemokines MCP-1 and macrophage inflammatory protein-1alpha.     

Allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in chronic skin lesions of atopic dermatitis

The majority of allergen-specific T cells derived from inhalant allergen patch test lesions in patients with atopic dermatitis were previously found to produce a restricted type-2 cytokine pattern. Recent studies, however, have revealed that in chronic eczematous skin lesions of patients with atopic dermatitis, expression of the type-1 cytokine interferon-gamma predominates. To evaluate cytokine production by allergen-specific T cells in chronic atopic dermatitis, we established house dust mite (Dermatophagoides pteronyssinus)-specific T-cell clones from the dermis of chronic skin lesions of sensitized adult patients with atopic dermatitis. Frequencies of skin-derived T cells proliferating in the presence of Dermatophagoides pteronyssinus were between one in 138 and one in 4255, indicating that only a minority of skin-infiltrating T cells are allergen specific. When these cells were analyzed for their capacity to produce interferon-gamma, the majority (71%) of these cells were found to express interferon-gamma mRNA and to secrete interferon-gamma protein, either alone or in combination with interleukin-4. Phenotypic analysis revealed that 15% of skin-infiltrating allergen-specific T cells were CD8+. No selection of Vbeta elements was detected in Dermatophagoides pteronyssinus-specific T-cell clones. These studies demonstrate that allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in lesional atopic dermatitis. The notion that the majority of allergen-specific, skin-infiltrating T cells are capable of producing interferon-gamma further supports the concept that interferon-gamma expression has major pathogenetic relevance for the chronic phase of atopic dermatitis.     

Effects of monocyte purification and culture on integrin expression

Selective alterations in the surface expression of members of the LeuCAM (leukocyte cell adhesion molecule) family of integrins occur during in vitro culture of human monocytes. Such changes may relate in part to cellular maturation, but also to activation following purification and culture of monocytes. In this paper, we examined the effects of monocyte isolation, adherence during culture and endotoxin exposure on the expression of these molecules and the ligand for LFA-1, ICAM-1 (CD54). Expressions of CD11b, CD18 and CD54, but not CD11a or CD11c, were higher on monocytes freshly isolated by density gradient separation and plastic adherence as compared with cells labelled directly in whole blood. However, the surface expression of the LeuCAMs and CD54 on cultured monocytes was not affected by short-term adherence to plastic for 2 h, as determined by comparisons of their expression on adherence-isolated and elutriated monocytes. In contrast, prolonged adhesion of monocytes for up to 21 days in culture altered expression of CD11a without affecting that of the other LeuCAMs or CD54. Expression of CD11a decreased more rapidly on adherence-maintained cells as compared with suspension-cultured cells. Our results show that cellular manipulations required for in vitro studies of monocyte/macrophages may alter expression of the LeuCAMs.     

Isolation of frog and chicken cDNAs encoding heparin cofactor II

In atopic eczema both in local inflammatory reactions and in peripheral blood high interleukin (IL) 4: interferon-gamma (IFN-gamma) production ratios have been demonstrated, indicating predominance of TH2 cell subsets resulting in increased IL-4 production and high serum IgE. The in vitro immunomodulatory effects of recombinant human soluble IL-4 receptor (rsIL-4R) on IL-4-stimulated lymphocyte proliferation, IgE and IFN-gamma production were studied in peripheral blood mononuclear cells from 10 patients with atopic eczema and seven healthy donors. In addition to control cultures (without any stimulus) and cultures with simultaneous application of rsIL-4R and IL-4, time-kinetic experiments were performed. We further investigated the influence of rsIL-4R on IL-4 production in staphylococcal enterotoxin B (SEB) stimulated peripheral blood mononuclear cells. Early addition of rsIL-4R to IL-4-stimulated peripheral blood mononuclear cells resulted in an increase in IFN-gamma production and in suppression of IL-4 induced proliferation and IgE secretion. Unexpectedly, rsIL-4R in combination with SEB exhibited an IL-4 protective effect with a significant increase in detectable IL-4 in the culture supernatants. The present data support the assumption that rsIL-4R might be a promising new immunomodulatory substance in the treatment of atopic eczema.     

Membrane localisation of a UDP-sugar hydrolase, in Salmonella, is by an uncleaved N-terminal signal peptide

Many immunologic aspects of atopic dermatitis have been studied, but basic pathobiologic mechanisms of this disease remain unknown. In this study, we measured the production of interleukin-6 (IL-6) by peripheral blood T cells and monocytes from patients with atopic dermatitis in comparison to normal control subjects and patients with chronic psoriasis. We found that peripheral blood T cells isolated from patients with atopic dermatitis produced significantly higher levels of IL-6 (36.1 +/- 5.1 units/ml, n = 22) than T cells derived from either normal subjects (12.6 +/- 1.9 units/ml, n = 22) or patients with chronic psoriasis (26.7 +/- 4.1 units/ml, n = 7). T-cell activation was also measured in the patients with atopic dermatitis by soluble serum IL-2 receptor levels and were found to be significantly higher (623.7 +/- 8.1 units/ml, n = 8) than normal subjects (357.2 +/- 26.0 units/ml, n = 8). In contrast to the increased production of IL-6 by T cells in atopic dermatitis, there was no significant difference in the IL-6 production by peripheral blood monocytes derived from patients with atopic dermatitis compared to normal subjects. Thus, peripheral blood T cells derived from patients with AD spontaneously produce increased amounts of IL-6 compared to T cells from normal subjects, which may reflect the increased activation state of T cells in atopic dermatitis. These data support the concept that activated T cells or subsets of T cells may be important effector cells in mediating inflammatory activity in atopic disease.     

CD4+ type 1 and CD8+ type 2 T cell subsets in human leishmaniasis have distinct T cell receptor repertoires

The mechanism of protective immunity and immunologic resistance against intracellular pathogens is believed to involve the activation of Ag-specific T cells. The T cells involved in protection/resistance to Leishmania can be studied using localized American cutaneous leishmaniasis (LCL) as a model, because the disease is often self-healing. Our study was undertaken to identify specific T cell populations that had accumulated in LCL lesions on the basis of TCR V beta gene usage. RNA was derived from skin lesions and blood of eight LCL patients, as well as from purified CD4+ and CD8+ subsets from the lesions and blood of three patients. After synthesis of cDNA, V beta gene usage was assessed by polymerase chain reaction. In all eight patients, several V beta gene families were overrepresented in lesions compared to blood. More importantly, the TCR V beta repertoires of both lesional CD4+ and CD8+ subsets were skewed compared to the repertoire of the respective subsets in the blood of the same donor. The overrepresented V beta s in the CD4+ and CD8+ subsets from lesions were in most instances disparate, particularly with the V beta 6 TCR skewed in the lesional CD8+ subset. Not only were the TCR repertoires of the overrepresented V beta in the lesional CD4+ and CD8+ subsets generally distinct, but the cytokine mRNA expressed by these subsets were also discrete. Strikingly, the CD4+ subset was characterized by IFN-gamma mRNA expression and the CD8+ subset by IL-4 and IL-10 mRNA expression. These data indicate that the pathogenesis of human leishmaniasis may be explained by the balance of CD4+ type 1 and CD8+ type 2 T cells, which probably recognize distinct sets of Ag.