The human erythrocyte receptor for the major activation fragments of the third and fourth components of complement (HuE-C3bR) was isolated from individual donors. Erythrocytes were surface labeled with 15I and solubilized in Nonidet P-40. HuE-C3bRwas purifiedbyusingC3-Sepharose affinitychromatography and analyzed by autoradiography of NaDodSO4/ polyacrylamide gels. Three distinct receptor patterns were demonstrated. Type a had a single major band with Mr of 190,000, type b had a single major band with Mr of 220,000, and type c had two major bands of Mr 190,000 and 220,000. In all three types, a minor band accounting for <25% of the total radioactivity was usually observed at a Mr 15,000 greater than that of each major band. Identical autoradiographic patterns were obtained by affinity chromatography using methylamine-inactivated C4-Sepharose or by immunoprecipitation of solubilized membranes with a monoclonal antibody against HuE-C3bR. All three types were distinct after reduction and alkylation, although the apparent Mr uniformly increased by ""30,000. Characterization of HuE-C3bR types in 33 unrelated individuals demonstrated that 23 had type a, 1 had type b, and 9 had type c. Family studies provide evidence for transmission by two codominant alleles. Thus, in the normal population two alleles appear to control expression of HuE-C3bR phenotypes and account for the polymorphism of this integral membrane glycoprotein.The human erythrocyte receptor § for the major activation fragments of the third (C3b) and fourth (C4b) components of complement (HuE-C3bR) has been previously isolated by sequential chromatography of pooled donor membranes. It was initially characterized on NaDodSO4/polyacrylamide gels as a single glycoprotein with a Mr of ==205,000 (2-5). Polyclonal antibodies against this glycoprotein inhibited immune adherence (2). This glycoprotein also accelerated decay of the alternative (2) and classical (4) pathway C3 convertases. Using a modified cell surface labeling procedure (6) and affinity chromatography, we previously reported the isolation of a rabbit alveolar macrophage receptor for C3b (7) and characterized its ligand binding specificity (8). Extending this method to human erythrocytes allowed us to isolate the C3b receptor from individual donors. We now report previously unrecognized polymorphism of this integral membrane glycoprotein. MATERIALS AND METHODS Surface Labeling and Solubilization of Human ErythrocyteMembranes. Approximately 30 ml of human blood was collected from each donor in 1.5 ml of 100 mM EDTA or in 4.5 ml of citrated dextrose (Fenwal Laboratories, Deerfield, IL). Blood was centrifuged at 800 x g for 8 min at 4°C, and the plasma and buffy coat were removed. The packed human erythrocytes were then washed at 4°C three times in phosphate-buffered saline (10 mM potassium phosphate/150 mM NaCi, pH 7.40). After each centrifugation, a portion was removed such that approximately one-half of the original packed cell volume was retained. Immediately before iodination, cells were ...

Proteins binding the third component of complement (C3) were isolated by affinity chromatography from surface-labeled solubilized membranes of human peripheral blood cells and cell lines. The isolated molecules were subjected to NlttodSO4/PAGE, and autoradiographs of these gels indicpe. that C3-binding [1][2][3][4]. This molecule has a number of interesting structural and regulatory features (1, 2, 5-11), including an unusual polymorphism in which there is an -60,000 difference in Mr among the three recognized alleles (5-7). CR2, the C3d receptor of human peripheral blood cells, is an -140,000 Mr integral membrane glycoprotein found on human B lymphocytes (12, 13). It is probably identical to a B-cell antigen defined by the monoclonal antibody anti-B2 (12, 14) and a C3b-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.binding protein purified from Raji cells (15). CR2 apparently binds to the C3d fragment within intact C3b (15).Our laboratory has employed affinity chromatography to isolate CR1 (5,7,8) and CR2 (16), as well as a C3b-specific integral membrane glycoprotein from rabbit alveolar macrophages (17, 18). In our studies of CR1 and CR2 on human leukocytes, we routinely observed a third group of molecules with Mrs of 45,000 to 70,000 (gp45-70) with binding specificity for C3. This report represents our initial evaluation of these C3-binding proteins of human leukocytes, including their isolation, cell distribution, electrophoretic mobility, and specificity for C3 and its proteolytic fragments. MATERIALS AND METHODSIsolation of Human Peripheral Blood Cells. Normal individuals donated "30 ml of blood for preparation of erythrocytes (E) or up to 500 ml for preparation of leukocytes. E were washed in phosphate-buffered saline (Pi/NaCl; 10 mM potassium phosphate/150 mM NaCl, pH 7.40) and resuspended in a 10% suspension. Mononuclear and polymorphonuclear (PMN) cell populations were separated by dextran sedimentation followed by Ficoll/Hypaque centrifugation (5, 8). Cell populations enriched for T lymphocytes, B plus null lymphocytes, or monocytes were prepared and analyzed as previously described (19). Greater than 80% of the monocyte-enriched population was esterase positive. Analysis of cell surface characteristics for the enriched lymphocyte populations showed that >75% of the T-cell-enriched population formed E rosettes with only 1% possessing surface immunoglobulin, and >60% of the B plus null cells had detectable surface immunoglobulin with <5% forming E rosettes.Cell Lines. The Raji cell line (an Epstein-Barr-virus-negative B-lymphoid line), the GM3104, GM3161, and GM3164 cell lines (all Epstein-Barr-virus-transformed B-cell lines), and the Jurkat T-cell line were grown in T-150 flasks at 37°C in a 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% fetal bovine serum (Sterile Systems, Logan, UT), 4 mM glutamine, and an antibioti...

In 161 ambulatory rheumatic disease patients receiving long-term prednisone therapy, diaphyseal mass (DM) and metaphyseal mass (MM) of the forearm were measured by single photon absorptiometry, and bone radiographs were reviewed when available. Multivariate analysis of treatment and patient characteristics demonstrated that glucocorticoid-induced osteopenia (defined as an elevated DM:MM ratio) and bone fractures occurred with similar frequency in patients of each sex, in whites and blacks, in patients with various rheumatic diseases, and in patients receiving different regimens of prednisone therapy. However, large cumulative doses of prednisone were associated with elevated DM:MM ratios as well as with bone fractures, and menopause or age 2250 years (males or females) was associated with bone fractures. We conclude that longterm therapy with various prednisone regimens results in glucocorticoid-induced osteopenia and fractures. This affect is cumulative, occurs in all patient groups, and results in more bone fractures in certain groups.It has long been recognized that long-term administration of oral glucocorticoids can result in a significant loss of bone mass (1-3). The degree of loss is more severe in areas of the skeleton with a high content of trabecular bone, such as vertebrae and ribs. It is less striking in the diaphyses of long bones, which consist primarily of compact cortical bone (4-6).We have previously used single photon absorptiometry to measure appendicular bone mineral mass in normal individuals and rheumatic disease patients (7-10). Measurements at diaphyseal and metaphyseal sites of the radius demonstrate a constant relationship between bone mineral mass at the diaphyseal site and that at the metaphyseal site, both in normal individuals and in most patients with conditions associated with osteopenia (for example, idiopathic osteoporosis). However, in osteopenia associated with glucocorticoid therapy, there is a greater loss of metaphyseal mass (MM) than diaphyseal mass (DM), and the DM:MM ratio increases (7-10). A combination of factors may account for this observation. First, the metaphyseal site contains a higher proportion of trabecular bone (>25%) than the diaphyseal site (-5%) (1 1). Second, the metaphyseal site has a greater surface area than the diaphyseal site, and endosteal resorption of cortical bone may be more easily measured at the metaphyseal site.Since measurement of DM:MM ratios provides a simple, inexpensive, and rapid method of identifying patients with glucocorticoid-induced osteopenia, we have measured these ratios in 161 patients with various rheumatic diseases treated with long-term glucocorticoid therapy. This report evaluates whether different glucocorticoid treatment regimens and patient characteristics are associated with glucocorticoid-induced osteopenia and bone fractures.

The human receptor for C3b and C4b (C3bR) t was initially isolated from pooled donor erythrocyte membranes (1). Although characterized as an integral membrane glycoprotein of ~200,000 daltons on SDS-polyacrylamide gels (1-4), recent studies have demonstrated that C3bR is polymorphic (5-7). In reports from our laboratory, autoradiographs of purified surface-labeled C3bR from erythrocytes (E) in 33 normal donors demonstrated three distinct patterns (5). ~70% of individuals had a single major band of -190,000 daltons (C3bR-AA), ~3% of individuals had a single major band of -220,000 daltons (C3bR-BB), and the remaining ~27% of individuals had both major bands (C3bR-AB). Family studies provided evidence that two codominant alleles regulated these major bands (5). Under this model, C3bR-AB individuals were heterozygous for the two alleles, A and B, and C3bR-AA or C3bR-BB individuals were homozygous for their respective alleles. Although the C3bR phenotype found on E was also present on other C3bR-bearing cells in the same individual, several cell-specific structural differences were also identified. First, on polymorphonuclear cells (PMN), the Mr of C3bR was ~5,000 daltons greater than that on E (6). Second, on E, less intense minor bands co-purified with C3bR and were found ~ 15,000 daltons above major bands on autoradiographs (5, 6). These minor bands, found only on E, accounted for <25% of the total C3bR. The percent represented by this minor band differed among individuals but was characteristic for a given individual. Third, among C3bR-AB individuals, vastly different (up to 30-fold) amounts of either the ~190,000-or ~220,000-dalton C3bR molecules were found on E and peripheral blood leukocytes (5, 6). The factors responsible for this variable expression among heterozygous individuals are unknown.In a study from another laboratory, this same polymorphism was described in a larger group of 111 unrelated individuals (7). However, as our laboratory has isolated C3bR from additional normal individuals we have found several novel

Twenty-three rheumatic disease patients with glucocorticoid-induced osteopenia (defined by measurement of forearm bone mass) completed an 18-month double-blind, randomized study to assess the effect of oral calcium and 1,25-dihydroxyvitamin D ( 1,25-OH2D) or calcium and placebo on bone and mineral metabolism. Intestinal 47Ca absorption was increased (P < 0.05) and serum parathyroid hormone levels were suppressed (P < 0.01) by 1,25-OH2D (mean dose 0.4 pgl day); however, no significant gain in forearm bone mass occurred, and bone fractures were frequent in both groups. In the 1,25-OH2D group, histomorphometric analysis of iliac crest biopsy specimens demonstrated a decrease in osteoclasts/mm2 of trabecular bone (P < 0.05) and parameters of osteoblastic activity (P < 0.05), indicating that 1,25-OH2D reduced both bone resorption and formation. We conclude that 1,25-OH2D should not be used for treatment of glucocorticoidinduced osteopenia. Since patients receiving calcium and placebo did not exhibit a loss of forearm bone mass, elemental calcium supplementation of 500 mg daily might be useful to maintain skeletal mass in patients receiving long-term glucocorticord therapy. Submitted for publication March 27, 1984; accepted in revised form July I I , 1984. Oral administration of glucocorticords in humans results in severe osteopenia caused by several mechanisms (1-3). Histologic studies of bone in humans receiving glucocorticoids demonstrate decreased bone formation (4-6) and increased numbers of osteoclasts and osteoclast-resorbing surfaces (5-7). The decrease in formation rate probably represents a direct effect of glucocorticoids on osteoblasts. Specifically, glucocorticoids decrease the recruitment of progenitor cells to osteoblasts and the synthesis of collagen and noncollagen protein by preexisting osteoblasts (8,9).The mechanism by which glucocorticoids increase bone resorption is more controversial. In vitro, glucocorticoids inhibit osteoclast activity (3, lo). However, in vivo, glucocorticoids greatly decrease intestinal calcium absorption (2,11,12) and may stimulate parathyroid hormone (PTH), thus providing an indirect stimulus to osteoclastic activity (6,13