Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN)

Elucidation of the interactions between C5a and granulocytes is central to understanding the role of C5a in inflammation. In this study, interactions between C5a and PMN have been studied at two levels. Binding of human C5a to intact human cells has been characterized by using the radiolabeled ligand 125I-C5a. Binding is shown to be reversible, saturable, and to reach equilibrium in 60 to 90 min at 0 degrees C. Results show high affinity C5a binding sites with Kd = 2 X 10(-9) M and a range of 50,000 to 113,000 binding sites per PMN. These values for C5a receptors are comparable with the number of fMLP and LTB4 receptors on PMN. Binding of C5a to PMN fails to reach equilibrium at 37 degrees C because there is an irreversible loss of available surface receptors caused by an active internalization of the ligand-receptor complex. Interactions between C5a and human PMN were characterized further by cross-linking experiments, with the use of ethylene glycol bis succinimidylsuccinate (EGS). Cross-linking of 125I-C5a to intact PMN followed by subcellular fractionation revealed a single radioactive band present only in the plasma membrane fraction and visualized by autoradiography. Similar experiments resulted in a covalent linkage between 125I-C5a and a component in the isolated plasma membrane of PMN. The covalent complex containing C5a and a putative receptor has been visualized by autoradiography as a single 60,000 Mr complex on SDS-PAGE. The complex is not present when experiments are performed in the presence of excess unlabeled C5a or in the absence of EGS. Therefore, the putative receptor for C5a on human neutrophils is estimated to be approximately 48,000 Mr, assuming contribution of 12,000 to 13,000 daltons by the ligand 125I-C5a.     

Interaction of human monomeric C3b with its receptor (complement receptor type 1, CR1) on neutrophils. Evidence for negative cooperativity

The binding of highly purified monomeric 125I-C3b to its receptor (CR1) on resting human polymorphonuclear neutrophils (PMN) was analyzed under equilibrium conditions, at 4 degrees C and low ionic strength. Scatchard analysis of specific binding data yielded curvilinear concave upward plots, which resulted from the presence of site-site interactions of the negative type among PMN C3b-receptors (negative cooperativity), as shown by dissociation kinetic experiments. Indeed, the dissociation rate of 125I-C3b from PMN was markedly increased in the presence of an excess of unlabeled C3b in the dilution medium and was directly dependent on the degree of initial receptor occupancy with the radioligand. These interactions occurred when 2% of the receptors were occupied with 125I-C3b and resulted in a 4-fold decrease in CR1 affinity when the receptor went from its "empty" to its "filled" conformation. In a disease associated with a continuous production of C3b (factor I deficiency), CR1 on in vivo circulating PMN was found to be in a "low affinity" and "high dissociating" state similar to that of normal CR1 at high occupancy. Finally, negative cooperativity among CR1 sites disappeared after PMN activation with chemotactic peptides.     

Characterization and affinity cross-linking of receptors for human recombinant lymphotoxin (tumor necrosis factor-beta) on a human histiocytic lymphoma cell line, U-937

Recombinant human lymphotoxin (rhLT) expressed in a mammalian cell line was purified and used to examine its receptors on the human histiocytic lymphoma cell line U-937. rhLT was radioiodinated by the IODO-GEN method to a specific activity of 60 microCi/micrograms; the labeled protein was biologically active in the cytolytic assay, and displaceable binding to U-937 cells was observed. The specific binding reached a plateau within 10, 60, and 180 min at 37, 23, and 4 degrees C, respectively. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 0.6 nM and a capacity of 33,000 +/- 7,000 binding sites/cell. The binding of 125I-rhLT to U-937 cells could be inhibited by excess unlabeled rhLT or recombinant human tumor necrosis factor (rhTNF), suggesting a common receptor for both molecules. As competitive inhibitor of the binding, rhTNF was equal in its potency to rhLT. Bacterial derived rhLT lacking carbohydrate was also found equipotent to cell line-derived rhLT for cell binding, indicating that carbohydrate plays no significant role in receptor interaction. Additionally, 125I-rhLT was covalently attached to the cell surface via a bifunctional cross-linking reagent, ethylene glycol bis(succinimidyl succinate), solubilized, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cross-linking of the receptor to rhLT revealed two distinct bands at approximate molecular masses of 100,000 and 120,000 daltons. Both bands were absent when unlabeled rhLT or rhTNF was used for competition, indicating the specificity. Affinity cross-linking of U-937 cells with 125I-rhTNF, however, provided only a single band with a molecular mass of about 100,000 daltons. These results suggest that the manner in which rhLT interacts with its receptor is perhaps somewhat different from that of rhTNF.     

Identification of the receptor for atrial natriuretic factor on cultured vascular cells

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.     

Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein

The receptor for vasoactive intestinal peptide (VIP) was identified in rat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis(propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80,000 and 56,000 daltons (Da). Both the large and small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 degrees C for 30 min with 1 microgram/mL chymotrypsin, trypsin, or elastase to fragments that that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80- and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (approximately 50%). The WGA-bound fraction (eluted with N-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80- and 56-kDa species to endo-beta-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydrate chains. The receptor species do not appear to have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-beta-acetylglucosaminidase H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a receptor for C5a anaphylatoxin on human eosinophils

The complement anaphylatoxin peptide C5a is well known to activate human polymorphonuclear leukocytes through receptor-mediated processes. C5a has also been reported to activate eosinophils for both chemotaxis and hexose uptake. We characterized the receptor molecule for human C5a on human eosinophils and compared it with the receptor on human neutrophils. At 4 degrees C, uptake of 1 nM 125I-C5a reaches equilibrium within 10 min on both cell types. Binding of 125I-C5a occurs over a concentration range comparable to that which stimulates lysosomal enzyme release and hexose uptake in both cell types. Scatchard analyses of the data indicate the presence of two receptor populations on eosinophils; a high affinity receptor with 15,000-20,000 sites/cell and a Kd of 3.1 +/- 0.6 x 10(-11) M, and a low affinity receptor with approximately 375,000 sites/cell and a Kd of 1 x 10(-7) M. Parallel experiments with neutrophils indicate the presence of a single receptor population with approximately 90,000 sites/cell and a Kd of 4.8 +/- 0.1 x 10(-10)M. The eosinophil receptor molecule was further characterized by covalently cross-linking 125I-C5a to cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material. Autoradiography indicates the presence of a dominant C5a-eosinophil receptor complex with an apparent mass of 60-65 kDa. The corresponding neutrophil-C5a receptor complex has an apparent mass of 50-52 kDa as observed by others. When the cross-linked 125I-C5a-receptor complex was treated with cyanogen bromide, different patterns were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for neutrophils and eosinophils. Thus, human eosinophils have a receptor for C5a anaphylatoxin which appears to be distinct from the C5a receptor present on human neutrophils.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells

Erythroid differentiation factor (EDF), which is structurally related to transforming growth factor-beta family and induces differentiation of murine erythroleukemia cell clone F5-5, has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-EDF to F5-5 cells is time- and temperature-dependent, specific, saturable, and reversible. Transforming growth factor-beta 1 has no significant effects on growth of F5-5 cells and binding of 125I-EDF to F5-5 cells. Scatchard analysis of the binding data indicated that F5-5 cells have a single class of binding sites (3,200/cell) with an apparent Kd of 3.1 X 10(-10) M. Affinity cross-linking experiments demonstrated three radiolabeled components of 140,000, 76,000, and 67,000 daltons under both reducing and nonreducing conditions. Labeling of these three components has been inhibited by incubation of the cells with excess unlabeled EDF. These results imply molecular weights of 115,000, 51,000, and 42,000 for the EDF receptors on this cell line.     

Covalent cross-linking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts

125I-labeled vasoactive intestinal polypeptide (125I-VIP) was covalently cross-linked with its binding sites on intact cultured human lymphoblasts by each of three bifunctional reagents: disuccinimidyl suberate (DSS), ethylene glycol bis(succinimidyl succinate) (EGS), and N-succinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH). A fourth cross-linking agent with a shorter chain length, N-hydroxysuccinimidyl 4-azidobenzoate (HSAB), was much less effective in cross-linking 125I-VIP to the site. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately equal to 50,000 +/- 3,000, regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by 10(-6) M unlabeled VIP and was partially blocked by the homologous hormones secretin and glucagon. The relative potencies of these peptides in blocking the cross-linking of 125I-VIP to the Mr approximately equal to 50,000 band of the lymphoblasts (VIP greater than secretin greater than or equal to glucagon) were similar to those previously found for competitive inhibition of 125I-VIP binding to its putative high-affinity receptor on these cells. The covalent cross-linking required a bifunctional reagent; it was dependent on both the number of Molt cells and the concentration of 125I-VIP. The apparent molecular weight of the cross-linked species was unchanged by treatment with dithiothreitol. These observations suggest that the Mr = 50,000 species represents 125I-VIP cross-linked to a specific plasma membrane receptor and that the receptor does not contain interchain disulfide bonds.     

Molecular characteristics of nerve growth factor receptors on PC12 cells

Cross-linking of 125I-nerve growth factor (NGF) to PC12 cells with the photoreactive heterobifunctional agent N-hydroxysuccinimidyl-4-azidobenzoate results in the labeling of two major bands with Mr 158,000 and 100,000 and a minor band with Mr 225,000 as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Binding of 125I-NGF to and cross-linking into all these species is abolished in the presence of excess unlabeled NGF but not in the presence of unlabeled epidermal growth factor, insulin, or bovine pancreatic trypsin inhibitor. When PC12 cells with bound 125I-NGF are incubated in excess unlabeled NGF at 0 degree C prior to cross-linking, only the Mr 158,000 species remains. In addition, binding of 125I-NGF to the Mr 158,000 complex is trypsin-resistant, whereas binding to the Mr 100,000 complex is not. These experiments identify the Mr 158,000 species as the slow NGF-receptor complex (chase stable at 0 degree C) and the smaller Mr 100,000 species as the fast NGF-receptor complex (trypsin sensitive). Furthermore, 125I-NGF bound to the former but not to the latter species is displaced by very-low concentrations of NGF, showing that at least a significant fraction of the high-molecular-weight slow receptor is also a high-affinity receptor. This identification is supported by the finding that chick sensory neurons which possess both high- and low-affinity receptors exhibit two major labeled bands with Mr 145,000 and 105,000 as a result of cross-linking with 125I-NGF, whereas a cell population enriched in non-neuronal cells, which possess only low-affinity receptors, exhibits only the Mr 105,000 component. A shift in molecular weight of both species after pretreatment with neuraminidase indicates that both complexes contain sialoglycoproteins and rules out the possibility that differences in sialic acid content are responsible for the difference in molecular weight of the two complexes. The relative amount of the labeling of these two complexes is not affected by the presence of protease inhibitors nor by a variation of 5000-fold in cross-linker concentration. These results place some limits on possible models for the NGF receptors and their interconversion.     

Identification of an antigenic epitope and receptor binding domain of human C5a

Nine different murine anti-human C5a monoclonal antibodies have been produced and characterized. They exhibit Kas for the 125I-labeled ligand that range from 0.4 to 48 X 10(8) M-1, and they display limited cross-reactivity with C5a from other species. Each of these antibodies has been found to compete with the granulocyte C5a receptor for binding site(s) on the C5a polypeptide. Exploration of the antigenic topography of C5a revealed that the immunodominant portion of this glycopolypeptide resides between residues Lys20 and Arg37, with the area surrounding Cys27 being particularly important. In addition, a specific C5a derived tryptic peptide containing these amino acid residues competes with 125I-C5a for binding to the receptor. These observations are consistent with previously published data and suggest that this area of the C5a molecule is an important part of the receptor "recognition domain", and thus plays a critical role in the C5a receptor interaction.     

Characterization of C1q receptor expression on human phagocytic cells: effects of PDBu and fMLP

The receptor-mediated binding of C1q to human phagocytic cells was investigated in this study. By using a C1q binding assay, we determined that purified, elutriated monocytes expressed an average of 4.6 X 10(5) C1q receptors (C1qR) per cell, with an equilibrium binding constant (Keq) of 0.91 X 10(7) (M-1). When analyzed in an identical manner, the polymorphonuclear leukocytes (PMN) expressed an average of 4.2 X 10(5) C1qR per cell, with a Keq for C1q of 1 X 10(7) (M-1). Fluorescent flow cytometric analysis showed that C1q was bound by 98% of the monocytes studied. Further, the pattern formed by these cells was consistent with a normal log distribution, indicating that this was a homogeneous population of cells. When PMN were assayed with flow cytometry, however, we found that C1q was bound by an average of only 45% of the PMN analyzed. Further, these PMN were not dispersed in a normal log distribution, indicating some heterogeneity among the cells that bind C1q. We examined the abilities of the chemoattractant N-formylmethionylleucylphenylalanine (fMLP) and the phorbol ester phorbol dibutyrate (PDBu) to modulate expression of C1qR as compared to the receptor for C3b (CR1). Pretreatment of the monocytes and the PMN with either 10(-6)M fMLP or 10 ng/ml of PDBu significantly increased cell surface CR1 expression, as reported previously by other investigators. In contrast, no significant increase in the number of C1qR on the monocytes or the PMN was observed with any of the concentrations of fMLP or PDBu used during pretreatment. However, with certain pretreatment doses of these agents, some reduction was noted in the amount of 125I-C1q bound to the monocytes and the PMN. This study characterizes the binding of C1q to purified monocytes and confirms previously published values for PMN. The distribution of cells expressing C1qR is shown to be significantly different between identically treated populations of monocytes and PMN. Finally, the abilities of fMLP and PDBu to modulate the binding of C1qR are examined. Our results indicate that the control of C1qR expression differs markedly from that of CR1.     

Identification of the bombesin receptor on murine and human cells by cross-linking experiments

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.     

Isolation and function of a human endothelial cell C1q receptor

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.     

Demonstration of a specific C3a receptor on guinea pig platelets

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin.     

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.     

Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity.     

Human platelets possess receptors for a lymphokine: demonstration of high specific receptors for HuIFN-gamma

This report demonstrates that 125I-recombinant human interferon-gamma (125I-rHuIFN-gamma) binds to high-affinity specific receptors on human platelets. Scatchard analysis of binding data indicates the presence of homogeneous sites estimated in the order of 150 to 200, with an apparent equilibrium dissociation constant, Kd, of 2 X 10(-10) M. The binding of 125I-rHuIFN-gamma to platelet membrane was inhibited by unlabeled rHuIFN-gamma but not by unlabeled rHuIFN-alpha or unlabeled rHuIFN-beta. High affinity binding sites for HuIFN-alpha were not detectable. Cross-linking of 125I-rHuIFN-gamma to platelet membrane proteins with the use of a bifunctional agent (DSS) yielded a predominant complex of 100,000 +/- 5,000 daltons on SDS-PAGE autoradiography, which confirms the presence of specific receptors for IFN-gamma. Two faint bands of lower m.w., 70,000 and 90,000, could also be visualized. Cross-linking of 125I-rHuIFN-alpha to platelet surface could not be demonstrated by using the same procedures. This is the first time that a receptor for a lymphokine (IFN-gamma) has been demonstrated on human platelets. These findings are consistent with data already published, suggesting an interrelationship between IFN and platelet function.     

Purification and partial characterization of rat ovarian lutropin receptor

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea and mouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.