(1/1480) Expression of the cell adhesion molecules on leukocytes that demarginate during acute maximal exercise.
The pulmonary vascular bed is an important reservoir for the marginated pool of leukocytes that can be mobilized by exercise or catecholamines. This study was designed to determine the phenotypic characteristics of leukocytes that are mobilized into the circulation during exercise. Twenty healthy volunteers performed incremental exercise to exhaustion [maximal O2 consumption (VO2 max)] on a cycle ergometer. Blood was collected at baseline, at 3-min intervals during exercise, at VO2 max, and 30 min after exercise. Total white cell, polymorphonuclear leukocyte (PMN), and lymphocyte counts increased with exercise to VO2 max (P < 0.05). Flow cytometric analysis showed that the mean fluorescence intensity of L-selectin on PMN (from 14.9 +/- 1 at baseline to 9.5 +/- 1.6 at VO2 max, P < 0.05) and lymphocytes (from 11.7 +/- 1.2 at baseline to 8 +/- 0.8 at VO2 max, P < 0.05) decreased with exercise. Mean fluorescence intensity of CD11b on PMN increased with exercise (from 10.2 +/- 0.6 at baseline to 25 +/- 2.5 at VO2 max, P < 0.002) but remained unchanged on lymphocytes. Myeloperoxidase levels in PMN did not change with exercise. In vitro studies showed that neither catecholamines nor plasma collected at VO2 max during exercise changed leukocyte L-selectin or CD11b levels. We conclude that PMN released from the marginated pool during exercise express low levels of L-selectin and high levels of CD11b. (+info)
(2/1480) Androgen influence on lacrimal gland apoptosis, necrosis, and lymphocytic infiltration.
PURPOSE: Previous studies have shown that ovariectomy and hypophysectomy cause regression of the lacrimal gland and have implicated androgens as trophic hormones that support the gland. The purposes of this study were to test the hypothesis that glandular regression after ovariectomy is due to apoptosis, to identify the cell type or types that undergo apoptosis, to survey the time course of the apoptosis, and to determine whether ovariectomy-induced apoptosis could be prevented by dihydrotestosterone (DHT) treatment. METHODS: Groups of sexually mature female New Zealand White rabbits were ovariectomized and killed at various time periods up to 9 days. Additional groups of ovariectomized rabbits were treated with 4 mg/kg DHT per day. At each time period, sham-operated rabbits were used as controls. Lacrimal glands were removed and processed for analysis of apoptosis as assessed by DNA fragmentation and for morphologic examination. DNA fragmentation was determined using the TdT-dUTP terminal nick-end labeling assay and by agarose gel electrophoresis. Labeled nuclei were quantified by automated densitometry. Sections were also stained for RTLA (rabbit thymic lymphocyte antigen), rabbit CD18, and La antigen. Morphology was evaluated by both light and electron microscopy. RESULTS: The time course of apoptosis exhibited two phases, a rapid and transient phase and a second prolonged phase. A transient phase peaked at approximately 4 to 6 hours after ovariectomy. The values for degraded DNA as a percentage of total nuclear area were 4.29%+/-0.79% and 4.26%+/-0.54%, respectively. The values for sham-operated controls examined at the same time periods were 1.77%+/-0.08% and 0.82%+/-0.21%, respectively. The percentage of degraded DNA at 24 hours after ovariectomy was not different from controls examined at the same interval after sham operation. The percentage of degraded DNA 6 days after ovariectomy was significantly increased (8.5%+/-2.4%), compared with sham-operated animals at the same time period (0.68%+/-0.03%). DNA laddering was more pronounced after ovariectomy. Dihydrotestosterone treatment in ovariectomized rabbits suppressed the increase in DNA degradation. Morphologic examination of lacrimal gland sections indicated that ovariectomy caused apoptosis of interstitial cells rather than acinar or ductal epithelial cells. Tissue taken 4 hours and 6 days after ovariectomy showed nuclear chromatin condensation principally in plasma cells. Increased numbers of macrophages were also evident. Significant levels of cell degeneration and cell debris, characteristic of necrosis, were observed in acinar regions 6 days after ovariectomy. Dihydrotestosterone prevented this necrosis. Increased numbers of RTLA+, CD18+, and La+ interstitial cells were also evident 6 days after ovariectomy. In addition, ovariectomy increased La expression in ductal cells. Dihydrotestosterone treatment prevented the increase in numbers of lymphoid cells and La expression. Dihydrotestosterone also promoted the appearance of mitotic figures in acinar cells and increased the sizes of acini by 43% (P < 0.05). CONCLUSIONS: Glandular atrophy observed after ovariectomy is likely to proceed by necrosis of acinar cells rather than apoptosis. This process begins with an apparent time lag after a rapid phase of interstitial cell apoptosis. These processes are accompanied by increased lymphocytic infiltration. These results suggest that a critical level of androgen is necessary to maintain lacrimal gland structure and function and that a decrease in available androgen below this level could trigger lacrimal gland apoptosis and necrosis, and an autoimmune response. Because apoptotic and necrotic cell fragments may be sources of autoantigens that can be processed and presented to initiate an autoimmune reaction, we surmise that cell death triggered by androgen withdrawal may trigger an autoimmune response such as that encountered in Sjogren's syndrome. (ABSTRACT TRUNCATED) (+info)
(3/1480) Antibodies to CD18 influence neutrophil migration through extracellular matrix.
Mac-1 (CD11b/CD18) is known to be involved in neutrophil (PMN) adhesion to endothelial cells and extracellular matrix. Although antibodies to CD 18 are being tested for therapy in humans, their role in PMN migration through the extracellular matrix is unknown. We used direct visualization to quantify PMN motility through reconstituted, three-dimensional gels of collagen type I. Gels were prepared with different concentrations of collagen (ranging from 0.1 to 1.0 mg/mL) and PMN migration was examined in the presence and absence of antibodies to CD18 (anti-CD18), with and without stimulation by N-formyl peptides. In low-concentration gels (<0.6 mg/mL), anti-CD18 had a significant influence on PMN migration, increasing motility in unstimulated PMN by 90% at 0.3 mg/mL collagen, and decreasing motility in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN by 70% at 0.4 mg/mL collagen. But antiCD18 had no effect on the rate of cell migration through high-concentration collagen gels (>0.6 mg/mL). PMN migration through collagen gels is CD18-dependent but only under conditions of high hydration, suggesting that CD18-mediated effects (e.g., adhesion to gel fibers) are only important when the fiber density is relatively low. Anti-CD18 inhibited, but did not eliminate, the adhesion of fMLP-stimulated PMN to the surface of collagen gels, suggesting that cells use multiple mechanisms for gaining traction within the gel. Because of the multiple modes of interaction between motile cells and the deformable fiber matrix, blockade of one component, such as CD18, can enhance the rate of cell migration under one set of conditions, and inhibit under another. (+info)
(4/1480) Characterization of beta2 (CD18) integrin phosphorylation in phorbol ester-activated T lymphocytes.
Integrins are transmembrane proteins involved in cell-cell and cell-extracellular-matrix interactions. The affinity and avidity of integrins for their ligands change in response to cytoplasmic signals. This 'inside-out' activation has been reported to occur also with beta2 integrins (CD18). The beta2 integrin subunit has previously been shown to become phosphorylated in T lymphocytes on cytoplasmic serine and the functionally important threonine residues after treatment with phorbol esters or on triggering of T-cell receptors. We have now characterized the phosphorylation of beta2 integrins in T-cells in more detail. When T-cells were activated by phorbol esters the phosphorylation was mainly on Ser756. After inhibition of serine/threonine phosphatases, phosphorylation was also found in two of the threonine residues in the threonine triplet 758-760 of the beta2 cytoplasmic domain. Activation of T-cells by phorbol esters resulted in phosphorylation in only approx. 10% of the integrin molecules. Okadaic acid increased this phosphorylation to approx. 30% of the beta2 molecules, assuming three phosphorylation sites. This indicates that a strong dynamic phosphorylation exists in serine and threonine residues of the beta2 integrins. (+info)
(5/1480) Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin.
Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that alpha5beta1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil beta1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the beta1 integrins. Antibodies or peptides that block alpha5beta1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block beta1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated beta1 integrins. (+info)
(6/1480) Monocyte activation in rheumatoid arthritis (RA): increased integrin, Fc gamma and complement receptor expression and the effect of glucocorticoids.
The aim of this work was to study the expression of beta 1- and beta 2-integrins, CR1, CD44 and Fc gamma receptors on peripheral blood monocytes in RA. The expression of these receptors was measured by flow cytometry, before and after treatment with low-dose prednisolone. Expression of the same receptors was also measured before and after treatment with metyrapone, a substance that inhibits the synthesis of cortisol in the adrenals. The expression of the beta 2-integrins CD11a, CD11b and CD18, of CD35 (CR1), and of Fc gamma RII and Fc gamma RI (CD32 and CD64) on monocytes was elevated in the RA patients compared with healthy controls, while the expression of the beta 1-integrins (CD29, CD49d, CD49f) was unaffected. A significant correlation between monocyte expression of CD64 and C-reactive protein (CRP), and blood platelet count, respectively, was found in the group of patients with RA. After 4-6 weeks of treatment with low-dose prednisolone, the expression on the monocytes of CD11a, CD11b, CD18, CD35, CD32 and CD64 was normalized. A significant correlation (r = 0.64, P = 0.02) was found between the decrease in expression of CD11b and clinical improvement after prednisolone treatment. Two days of metyrapone treatment, which significantly lowered the serum cortisol levels, elevated the expression of CD35 and CD49f. Priming of peripheral monocytes seems to be one of the mechanisms behind the recruitment of monocytes to the rheumatoid synovium. One reason for the good clinical effects of prednisolone in RA could be a down-regulation of adhesion and phagocytosis receptors on monocytes. (+info)
(7/1480) Specific activation of leukocyte beta2 integrins lymphocyte function-associated antigen-1 and Mac-1 by chemokines mediated by distinct pathways via the alpha subunit cytoplasmic domains.
We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (alphaMbeta2) but not lymphocyte function-associated antigen-1 (LFA-1; alphaLbeta2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1alpha in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1alpha were confirmed by expression of alphaM or alphaL in alphaL-deficient Jurkat cells. Moreover, expression of chimeras containing alphaL and alphaM cytoplasmic domain exchanges indicated that alpha cytoplasmic tails conferred the specific mode of regulation. Coexpressing alphaM or chimeras in mutant Jurkat cells with a "gain of function" phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the alphaL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of beta2 integrins. Our data suggest that a specific regulation of beta2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the alpha subunit cytoplasmic domains. (+info)
(8/1480) Lipopolysaccharide-coated erythrocytes activate human neutrophils via CD14 while subsequent binding is through CD11b/CD18.
Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP enhanced binding twofold, and this surplus was dependent upon CD14 but not on CR3. We conclude that ELPS interact with neutrophils via CD14, initially giving rise to cell activation; subsequently, binding is solely mediated by activated CR3. (+info)