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Although circulating phagocytic cells are important mediators of glomerular injury, their recruitment mechanisms are not completely understood. In this study, the intraglomerular trafficking of leukocytes was characterized in a rat model of acute glomerular injury induced by nephrotoxic serum (NTS). Polymorphonuclear (PMN) cells infiltrated, then disappeared rapidly, reaching a peak at 2 hr. By 6 hr the PMN migration had almost reversed but small numbers persisted until Day 7. The monocyte influx began almost simultaneously but was of lesser magnitude. However, the number of ED-1+ monocytes increased progressively from 60 min to reach a plateau by Day 2 and persisted to the end of the study (Day 28). Quantitation of intraglomerular Ia+ cells suggested in situ activation of monocytes within the glomeruli. Increased Ia+ cells were first evident on Day 2. By Day 5, 80% of the intraglomerular macrophages were Ia+. Complement depletion with cobra venom factor abrogated early albuminuria, delayed the initial PMN influx, but failed to attenuate monocyte migration. T lymphocytes appeared briefly between 10 min and 2 hr. In vitro proliferation study failed to demonstrate lymphocyte sensitization to glomerular basement membrane (GBM) antigens. A unique population of cells (OX19 OX8+), possibly representing natural killer cells, was present from Day 1 to Day 14. During the secondary wave of proteinuria (autologous phase), all leukocytes had disappeared except for macrophages and a small number of OX19-, OX8+ cells. A complex intraglomerular migration of leukocytes was triggered by the binding of nephrotoxic antibodies to GBM antigens. We speculate that this cascade involves several cell-to-cell interactions necessary for the full expression of glomerular injury.
In open-chest anesthetized dogs acute hypertension causes neutrophil and platelet adhesion to vascular endothelium and selectively potentiates constriction to serotonin in proximal coronary arteries. To examine underlying mechanisms, canine left anterior descending coronary arteries subjected to 15 min hypertension (LAD-HYP) and control left circumflex coronary arteries (CX) perfused at normal pressure were studied in organ chambers. In endothelium-intact LAD-HYP rings, constriction to serotonin was potentiated fourfold compared with control CX rings but was similar in denuded LAD-HYP and CX vessels. Endothelium-dependent relaxation to acetylcholine was not affected by acute hypertension. In LAD-HYP rings 10 microM LY 83583 (which depletes guanosine 3',5'-cyclic monophosphate and inhibits effects of endothelium-derived relaxing factor) augmented constriction to serotonin twofold. LY 83583 did not affect the serotonin response in hypertensive rings whose endothelium was mechanically removed. Blockade of either leukotriene D4 (LTD4) receptors (either with LY 171883 or SKF 102992) or thromboxane A2 (TxA2) receptors (with SQ 29548) partially blunted constriction to serotonin. Combined LTD4- and TxA2-receptor blockade completely normalized serotonin-induced constriction in LAD-HYP rings. In preconstricted LAD-HYP rings, relaxations to serotonin were markedly impaired but were restored by addition of ketanserin. Normalization of relaxation to serotonin in hypertensive vessels by ketanserin is likely due to inhibition of 5-hydroxytryptamine2 (5-HT2) receptors on platelet membranes. In conclusion, augmented constriction to serotonin in canine epicardial vessels exposed to acute hypertension is not due to an impairment of endothelium-dependent relaxation to the amine but to concomitant release of leukotrienes and TxA2 from leukocytes and platelets adhering to damaged endothelium. Activation of 5-HT2 serotonergic receptors on platelet membranes could be a possible trigger mechanism.
Therapeutic doses of glucocorticoids are thought to inhibit prostaglandin and leukotriene formation in humans. Several studies in animals, however, have failed to demonstrate modulation of eicosanoid biosynthesis by steroids in vivo. We administered prednisone (60 mg/day) to eight healthy volunteers and measured eicosanoid formation by a variety of cell types in vivo and ex vivo, using sensitive and specific physicochemical assays. We found that the in vivo course of prednisone failed to inhibit the synthesis of thromboxane A2, prostaglandin I2 (prostacyclin), prostaglandin E2, and leukotriene E4 in vivo and of leukotriene B4 ex vivo. Biosynthesis of leukotriene B4, thromboxane B2, and prostaglandins F2 and E2 by macrophage-rich bronchoalveolar lavage cells was strongly suppressed. These findings indicate that therapeutic regimens of glucocorticoids suppress eicosanoid biosynthesis in human macrophages but not in a number of other cell types with steroid receptors, the capacity for eicosanoid formation, and lipocortin-like material.
Chronic dialysis patients have several indices of immune deficiency. We examined the hypothesis that the biocompatibility of dialysis membranes may influence the ability of lymphocytes to express interleukin-2 (IL-2) receptors on their surface, a key event in cellular immune response. We investigated the potential role of the dialysis membrane in eight chronic hemodialysis patients. The study design was a cross-over study using cuprophane and polymethylmethacrylate (PMMA) membranes. Chronic dialysis with new cuprophane membrane leads to an increase in baseline expression of the two subunits of IL-2 receptors. IL2R alpha (p55, CD25) and IL-2R beta (p70), in peripheral blood mononuclear cell (PBMC). However, Phytohemagglutinin (PHA) stimulation of PBMC harvested after two weeks of dialysis with cuprophane membrane showed a markedly decreased expression of high affinity IL-2 receptors. These findings are reversed when patients were dialyzed with a PMMA membrane which is also associated with minimal complement activation. The increased expression of IL-2 receptor subunits are reproduced in vitro by direct contact of PBMC with cuprophane membrane and by the addition of the anaphylatoxin C5a. This study confirms the participation of lymphocytes in the complex blood-membrane interactions that occurs during dialysis; the results may be relevant to observations of immune deficiency in dialysis patients.
To test the hypothesis that mononuclear cells are stimulated to release interleukin 1 (IL-1) by bone fragments released in the bone microenvironment during the remodeling cycle, we have investigated the effects of bone matrix and some of its constituents on IL-1 secretin from peripheral blood mononuclear cells (PBMC). Increases in IL-1 activity were observed when either PBMC or adherent monocytes, but not lymphocytes depleted of monocytes, were co-cultured with either human or rat bone particles but not with latex particles of similar size. Co-culture of PBMC with bone particles in a transwell system where the cells were physically separated from the bone particles, or with osteoblast- or osteoclast-covered bone particles, did not stimulate IL-1 release, indicating that a physical contact between PBMC and the bone surface is required for eliciting IL-1 release. This was confirmed by the finding of a lower stimulatory effect of bone particles pretreated with etidronate, a bisphosphonate which decreases the bone binding capacity of PBMC. Constituents of bone matrix, such as collagen fragments, hydroxyproline, and, to a lesser extent, transforming growth factor-beta, but not osteocalcin, alpha 2HS glycoprotein, fragments of either bone sialoprotein or osteopontin, and fibronectin, stimulated PBMC IL-1 release in a dose-dependent fashion. Collagen-stimulated IL-1 release was partially and specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1-integrin cell surface collagen receptor. These data demonstrate that products of bone resorption, known to be chemotactic for mononuclear cells, stimulate PBMC IL-1 activity. These findings may help explain previous documentation of increased IL-1 secretion by circulating monocytes obtained from patients with high turnover osteoporosis.
17 alpha-Hydroxylase deficiency is characterized by defects in either or both the 17 alpha-hydroxylase/17,20-lyase activities. We have, for the first time, elucidated the molecular basis of the deficiency in a male pseudohermaphrodite with ambiguous external genitalia resulting from partial combined deficiency of both activities. The patient is found to be a compound heterozygote, carrying two different inherited mutant alleles in the cytochrome P45017 alpha (CYP17) gene. One allele, from his mother, contains a stop codon (TGA) in place of arginine (CGA) at amino acid position 239 in exon 4. Because this occurs at the N-terminal side of the heme binding sequence, the putative resultant truncated protein is nonfunctional. The second allele, from his father, contains a missense mutation encoding the substitution of proline (CCA) by threonine (ACA) at position 342 in exon 6. Reconstruction of this mutation by site-directed mutagenesis into human P45017 alpha cDNA followed by expression in COS 1 cells leads to the same amount of immunodetectable P45017 alpha protein as found with expression of the normal P45017 alpha cDNA, although both the 17 alpha-hydroxylase and 17,20-lyase activities are found to be reduced to 40-45% of those of the normal enzyme. The presence of ambiguous external genitalia in this 46 XY individual indicates that greater than 20% of the total normal 17,20-lyase activity is required for complete virilization in the male.
PBMC express cell surface receptors for extracellular matrix components known as integrins. We have recently shown that ligand binding to one PBMC integrin, the collagen receptor alpha 2 beta 1, stimulates the secretion of interleukin 1 (IL-1). We have now investigated the role of fibronectin (Fn), an adherence protein that has binding sites for both PBMC and collagen, in the generation of the IL-1 response to collagen. In contrast to collagen, Fn did not stimulate IL-1 release but Fn-depleted serum decreased the release of IL-1 induced by collagen. A polyclonal antiserum directed against Fn also decreased the collagen-induced IL-1 secretion. The IL-1 response to collagen from cells incubated in Fn-depleted serum was restored by the addition of either purified Fn or the 120-kD cell-binding fragment of Fn, which contains the cell-binding site but not the collagen-binding domain. Smaller Arg-Gly-Asp (RGD) peptides failed to enhance the PBMC response to collagen but inhibited in a concentration-dependent fashion the potentiating effect Fn. As expected, a MAb against the alpha 2 beta 1 collagen receptor decreased collagen-induced IL-1 release. However collagen-induced IL-1 release was also inhibited by a MAb against the alpha 5 beta 1 Fn receptor. The effect of the two MAbs was not additive, suggesting that the occupancy of both receptors by ligands is required in order for collagen to induce an maximal response from PBMC. The mechanism by which Fn exerts its effect remains unknown. However, flow-cytometric analysis revealed that Fn does not alter expression of the alpha2beta1 receptor on PBMC. These data demonstrate a potentiating effect of Fn on the collagen-induced secretion of IL-1 from human PBMC and suggest that this effect is mediated via the integrin alpha5beta1. These findings indicate a complex interactive role for specific integrin receptors in the regulation of the mononuclear cell immune response.
Epidermoid cervical carcinoma cells (CaSki line) have been established in continuous culture. When leukocytes from cervical cancer patients were incubated with CaSki culture fluid concentrates, inhibition of leukocyte migration was observed in more than 70 percent of the patients tested. By contrast, significantly less inhibition was observed with normal donor leukocytes or leukocytes from patients with other types of cancer. These results were consistent with the expression of tumor-associated antigen by CaSki cells. Analysis of the serum from the donor of the cell line at the time of tumor biopsy, and of CaSki culture fluids, demonstrated the presence of the beta subunit of human chorionic gonadotropin.