Granulocyte and monocyte adsorption apheresis (GCAP) for refractory skin diseases caused by activated neutrophils and psoriatic arthritis: evidence that GCAP removes Mac-1-expressing neutrophils.

In the present study, we have shown that granulocyte and monocyte adsorption apheresis (GCAP), an extracorporeal apheresis instrument whose column contains cellulose acetate (CA) beads, is useful for skin diseases attributable to activated granulocytes and psoriatic arthritis (PsA). We assessed the clinical effectiveness of GCAP and investigated the mechanisms underlying the adsorption of pathogenic granulocytes. The effect of GCAP was assessed in 14 patients with neutrophilic dermatoses and 16 with PsA. The mechanisms by which the instrument adsorbs activated granulocytes were investigated using an in vitro mini-column system that mimics the GCAP. Skin lesions and arthropathy improved in 22 of 29 patients (75.9%) and 14 of 18 (77.8%), respectively. Mac-1 (CD11b/CD18) expression on the peripheral neutrophils, increased compared with normal subjects, was reduced by GCAP. In the mini-column system, CA beads adsorbed 50% neutrophils; and adsorption was inhibited significantly by treating plasma with EDTA and blood cells with antihuman CD11b monoclonal antibody. GCAP was useful for treating neutrophilic dermatoses and PsA. GCAP adsorbs Mac-1-expressing neutrophils to the CA beads by the binding of complement component (iC3b) on CA beads and CD11b expressed on activated neutrophils.

Effect of uremia and hemodialysis on soluble L-selectin and leukocyte surface CD11b and L-selectin.

Leukocyte Mac-1 (CD11b/CD18) and L-selectin (CD62L) are implicated in leukocyte adhesion to endothelial cells. In this study, L-selectin and CD11b expression on leukocytes and soluble L-selectin (sL-selectin) serum levels were investigated in 17 nondialyzed patients with chronic renal failure (CRF), in 28 chronic hemodialysis patients before hemodialysis (basal state), and in 32 healthy subjects. These parameters were also monitored during hemodialysis with cuprophane and cellulose diacetate membranes in a crossover study in five patients. Granulocytes from CRF patients displayed lower expression of L-selectin and higher expression of CD11b than granulocytes from healthy subjects. On the other hand, baseline expression of L-selectin and CD11b on leukocytes from hemodialysis patients did not differ from that of healthy subjects. In CRF and hemodialysis patients, sL-selectin levels were significantly lower than in healthy subjects. During hemodialysis, cuprophane membrane induced an upregulation of granulocyte CD11b, a decrease in granulocyte L-selectin, and an increase in sL-selectin serum levels. Conversely, cellulose diacetate caused only a transient increase in granulocyte CD11b and did not modify granulocyte L-selectin and sL-selectin serum levels. High CD11b and low L-selectin expression on granulocytes in CRF patients suggests an activation state, which was not found in hemodialysis patients at the basal state. The lack of activation in hemodialysis patients could reflect the elimination of a uremic toxin by dialysis or a loss of granulocyte responsiveness because of the repetitive stimulation by hemodialysis treatment. The low serum levels of sL-selectin in CRF and hemodialysis patients also suggest granulocyte dysfunction.

Aggressive and chronic periodontitis correlate with distinct cellular sources of key immunoregulatory cytokines.

BACKGROUND: Chronic periodontitis (CP) and aggressive periodontitis (AP) are inflammatory diseases and the main cause of dental loss in adults. We aimed to investigate the expression of adhesion molecules and the source of proinflammatory and anti-inflammatory cytokines in circulating mononuclear cells from patients with CP and AP. METHODS: Peripheral blood mononuclear cells from healthy controls and CP or AP patients were collected. The expression of the cell adhesion molecules CD11a and CD11b, and the cellular sources of interleukin (IL)-4, IL-10, IL-12, interferon-γ, and tumor necrosis factor-α by distinct subpopulations of circulating leukocytes were determined using flow cytometry. RESULTS: The expression of CD11a, but not CD11b, was significantly higher within the CD4(+) and CD8(+) T cells in CP and AP than in healthy controls. The frequencies of tumor necrosis factor-α-expressing CD4(+) T cells and CD14(+) cells were higher in AP and CP, compared to healthy controls, respectively. Moreover, the frequency of IL-10 expressing CD14(+) cells was higher in CP, but not AP, compared to healthy controls CD4(+) T cells committed to IL-4 production was higher in CP than in healthy controls. CONCLUSION: These results suggest the participation of CD11a in the pathogenesis of periodontal lesions and show distinct cellular sources of immunoregulatory cytokines in AP versus CP.

Mobilization of an intracellular glycoprotein (Mac-1) on monocytes and granulocytes during hemodialysis.

We studied the upregulation of the intracellular glycoprotein Mac-1 (CD11b/CD18, CR3) on monocytes and granulocytes during 36 bicarbonate hemodialyses in 12 patients who were randomly treated with Cuprophan (Cu), Hemophan (He) or Polysulfone (PS; low-flux) membranes. The degree of mobilization of this adhesion protein was related to changes in granulocyte and monocyte count, generation of C3a and production of interleukin-1 beta in plasma. Mac-1 expression on granulocytes was significantly higher after 5 and 15 min of Cu hemodialysis as compared to He or PS dialyses (p < 0.001) and correlated to changes in granulocyte count at 15 min (r = 0.62 and r = 0.76, p < 0.001). No differences in early Mac-1 mobilization on circulating monocytes was observed despite a decrease in cell count. Mac-1 expression on monocytes and granulocytes in the venous blood line at 180 min of treatment was significantly higher during Cu dialysis as compared to He and PS dialyses (p < 0.02 and p < 0.001, respectively). Early generation of C3a was higher in patients on Cu dialysis than in He or PS dialysis (p < 0.001) and correlated both to granulocytopenia (r = 0.45, p < 0.01) and to the subsequent increase in Mac-1 expression on granulocytes (r = 0.63, p < 0.001). An early increase in Mac-1 expression on monocytes was accompanied by an increase in plasma interleukin-1 beta later during dialysis (p < 0.05). Studies of Mac-1 expression during hemodialysis increased the sensitivity of biocompatibility measurements and correlated better than complement generation to changes in granulocyte count as it mediates adhesion to endothelial cells.

Cell surface receptor modulation on monocytes and granulocytes during clinical and experimental hemodialysis.

We studied the modulation of cell surface receptors related to cell adhesion (L-selectin and Mac-1) on monocytes and granulocytes during clinical (7 patients treated with cuprophan, Cu, and polysulfone, PS, membranes, n = 14) and experimental Cu and PS hemodialysis (n = 14). The objective was to compare cell surface receptor modulation in vivo when large subpopulations of cells are withdrawn from the circulating pool with the experimental model when cells are not sequestrated. The expression of Mac-1 and L-selectin on monocytes increased during clinical Cu dialysis (p = 0.024 and p = 0.0096, respectively) but remained stable during PS dialysis. On granulocytes, an inverse receptor modulation of Mac-1 and L-selectin was observed during clinical Cu dialysis but not during PS dialysis. Mac-1 was significantly higher and L-selectin lower on granulocytes after 15 min of clinical Cu as compared to PS dialysis (p = 0.001 and p = 0.0093, respectively). During experimental Cu dialysis, Mac-1 expression increased and L-selectin decreased markedly and continuously on both monocytes and granulocytes. The L-selectin/Mac-1 ratio on monocytes and granulocytes may be used as an index of the ability of leukocytes to adhere and to be recruited to an inflammatory focus. This ratio was significantly lower during clinical Cu as compared to PS dialysis (p = 0.0008 and p = 0.0015 respectively) indicating that the recruitment of leukocytes to infection foci may be precluded in patients on Cu membranes. Both monocytes and granulocytes showed significantly lower L-selectin/Mac-1 ratio during and after experimental Cu as compared to PS dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

5-oxo-6,8,11,14-eicosatetraenoic acid is a potent stimulator of L-selectin shedding, surface expression of CD11b, actin polymerization, and calcium mobilization in human eosinophils.

5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of arachidonic acid formed by the oxidation of 5-hydroxy-6,8,11, 14-eicosatetraenoic acid by a highly specific dehydrogenase. 5-oxo-ETE is a chemoattractant for both neutrophils and eosinophils. Although it is not as effective as leukotriene B4 (LTB4) and platelet-activating factor (PAF) in stimulating neutrophil migration, we found that it is considerably more active than these and a variety of other lipid mediators as an eosinophil chemoattractant. Moreover, low concentrations of 5-oxo-ETE appear to enhance the responsiveness of these cells to PAF. The objectives of the current investigation were to identify rapid responses induced in eosinophils by 5-oxo-ETE that might be related to the infiltration of these cells into tissues. We found that 5-oxo-ETE is more effective than PAF and LTB4 in inducing both L-selectin shedding and actin polymerization in human eosinophils, whereas PAF is the most active of these mediators in stimulating calcium mobilization. The complementary effects of 5-oxo-ETE and PAF on actin polymerization and calcium mobilization may explain their synergistic effect on eosinophil migration. 5-oxo-ETE and PAF were equipotent in stimulating the surface expression of the beta2-integrin CD11b, but were slightly less potent than LTB4. 5-oxo-ETE- induced actin polymerization was subject to homologous but not heterologous desensitization. It was not prevented by incubation of eosinophils with inhibitors of protein kinase C (staurosporine), mitogen-activated protein kinase kinase (PD98059), or phosphatidylinositol-3-kinase (wortmannin). In conclusion, 5-oxo-ETE is a potent activator of human eosinophils and may be an important regulator of tissue infiltration of these cells.

Monocyte and granulocyte CD11b/CD18, CD62L expression and sICAM-1 concentration in the interdialytic period.

We studied cell surface modulation of CD11b/CD18 and CD62L on monocytes and granulocytes, sICAM-1 concentrations and the responsiveness of cells to exogenous fMLP in patients in the intra- (0-4 h Cuprophan dialysis) and interdialytic period (5-28 h) and in healthy subjects (0-24 h). The high CD11b/CD18, low CD62L granulocyte phenotype occurred rapidly during dialysis. By contrast, CD62L increased on the subpopulation of monocytes in circulation initially during dialysis and CD11b/CD18 was mobilized much slower. In the interdialytic period, the CD62L/(CD11b/CD18) ratio was reduced up to 12 h after start of treatment on both monocytes and granulocytes. This ratio was significantly lower than in healthy subjects up to 8 h after start of treatment. The responsiveness of granulocytes to exogenous fMLP, in terms of CD11b/CD18 mobilization, was significantly reduced in patients during and after hemodialysis as compared to that on granulocytes obtained from healthy controls. Monocytes were more refractory to fMLP up to 4 h after dialysis. sICAM-1 was significantly increased in patients before dialysis as compared to controls and remained elevated and fairly stable throughout treatment and in the interdialytic period. The variation in the expression of adhesion molecules on monocytes and on granulocytes in the interdialytic period was not related to the presence of activating serum factors remaining in the circulation after treatment. Our findings emphasize the importance of including the interdialytic period in the evaluation of dialysis membrane biocompatibility, especially when effects on monocytes are of interest.

Biomaterial development for cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is dependent on materials foreign to the patient for its successful application. When blood comes into contact with these so-called biomaterials, an inappropriate inflammatory response, which can be life-threatening in some patients, may develop. The reason for this inappropriate activation of host defence mechanisms is not entirely clear, however a number of strategies have evolved over the years to minimize this unwanted sequelae of CPB. These strategies include surface coating of the materials of the circuit, using new materials thought to improve biocompatibility, and using a number of pharmacological interventions designed to suppress the inflammatory response. Recently, there has been some evidence which indicates that the plasticizer employed in the polyvinyl chloride (PVC) tubing of the CPB circuit may play a part in the development of the inflammatory response. The work described in this paper tends to support this thesis. These studies showed that by washing the plasticizer from the surface of the PVC tubing, the biocompatibility, as reflected in the upregulation of CD11b on the surface of neutrophils, was enhanced. Furthermore, the use of non-plasticized substitutes for PVC had a similar effect. The benefit from removing the plasticizer was similar to that gained from surface coating with heparin, one of the conventional approaches to reducing the inflammatory response to CPB.

The initial reactions of graphite and gold with blood.

The initial reactions of graphite and gold with blood were investigated by short-time exposure to capillary blood and detection of surface-adsorbed plasma proteins and cells with an immunofluorescence technique. Antibodies specific to fibrinogen, complement factors C1q and C3c, prothrombin/thrombin, von Willebrand factor, and platelet- and leukocyte-membrane antigens were used. The fluorescence intensity was quantitated by computer-aided image analysis. Fibrinogen was the most abundant plasma protein immobilized on either surface, and dense populations of platelets adhered to the protein layer. Complement factors and prothrombin/thrombin were found on the graphite surface, localized in fibrin clots or related to platelets. Platelets were activated (expression of selectin CD62) on both surfaces but more extensively so on the gold surface. Activation of polymorphonuclear granulocytes (PMNGs), measured as expression of integrin CD11b, was seen on both surfaces but with different kinetics. On the graphite surface, the CD11b expression was only transient whereas on gold it increased with time. Our data indicate that graphite is more thrombogenic than gold but less inflammatory.

Complement activation during blood sampling procedures alters the expression of CD11b/CD18 on human neutrophils.

BACKGROUND AND OBJECTIVES: Differences in blood sampling and separation techniques can affect the quantitative levels of activation markers on different leukocyte subsets. We examined the effect of two sampling procedures of EDTA blood on the quantitative levels of two markers, the CD11b/CD18 antigen and the EG2 epitope on intracellular eosinophilic cationic protein (ECP), in neutrophils and eosinophils, respectively. MATERIALS AND METHODS: Sample I was collected directly after completion of blood donation by an open technique and constant flow from the transfer tube directly into EDTA tubes. After sampling, the transfer tube was manually closed with a clamp. Sample II was collected 45 s later by the same technique by opening the clamp. RESULTS: We found a significantly (p < 0.01) higher expression of CD11b/CD18 on neutrophils collected by sampling procedure II than on those collected by sampling procedure I. In contrast, we did not find any difference in the intracellular ECP expression between sampling procedures I and II. To further explore the mechanisms for the observed upregulation of CD11b/CD18, fragments of a transfer tube were incubated with normal human serum (NHS) and heat-inactivated NHS (NHS56), respectively, for 60 min at +37 degrees C. Leukocytes from healthy blood donors were then incubated for 15 min at +37 degrees C with these serum preparations. The CD11b/CD18 expression was significantly higher (p < 0.01) on neutrophils incubated with transfer-tube-activated NHS compared with NHS alone. However, when leukocytes were incubated with transfer tube activated NHS56, no difference was observed compared with incubation with NHS alone. In addition, by using confocal laser scanning microscopy, we could identify complement (C3c) deposits on the inner surface of the transfer tube fragments incubated in NHS, but not in NHS56, CONCLUSIONS: The quantitative level of the activation marker CD11b/CD18 on neutrophils, but not the EG2 epitope on intracellular ECP in eosinophils is significantly increased by a slight modification of the blood sampling procedure. It is suggested that the observed upregulation of CD11b/CD18 is caused by complement activation within the transfer tube. The results emphasize the importance of in-house data on the effect of variations in sampling procedures, particularly when data from healthy blood donors are included in clinical studies.

Immobilized heparin inhibits the increase in leukocyte surface expression of adhesion molecules.

The effect of heparin coating of artificial materials on the surface expression of leukocyte surface molecules was assessed in blood from 9 donors in an experimental model of extracorporeal circulation. The leukocyte surface molecules CD11b, complement receptor type 3 (CR3), and CD45, leukocyte common antigen (LCA), on granulocytes and monocytes exposed to unmodified polyvinyl chloride tubes increased significantly compared with unmanipulated controls (p < 0.001) whereas a very modest and nonsignificant increase was observed in blood circulated in heparin-coated tubes. Accordingly, a highly significant reduction in expression of the 2 molecules was achieved by heparin coating (p < 0.001). The expression of CD11b and CD45 correlated with the degree of complement activation in plasma (rho = 0.6, p = 0.003). The expression of CD14 (endotoxin receptor) and CD16 (Fc gamma III receptor) was increased to the same extent in blood from unmodified and heparin-coated tubes (p < 0.02), and no correlation to complement activation was seen (rho = -0.275, p = 0.17 and rho = -0.004, p = 1, respectively). In conclusion, heparin coating of artificial surfaces has a substantial effect, reducing the expression of molecules involved in cellular adhesion and activation (CD11b and CD45), and the increase of these molecules by unmodified surfaces is complement dependent. In contrast, up-regulation of other surface molecules (CD14 and CD16) seems to be independent of heparin coating and complement activation and, thus, might be regulated by other mechanisms.