Characterization of the C1q receptor on a human macrophage cell line, U937.

The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies.     

Expression and function of gingival fibroblast C1q receptors are upregulated by interleukin-1β and transforming growth factor-β

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate-bound C1q with receptors for the collagen-like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL-1β and TGF-β increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration-dependent, saturable (20 ng/ml IL-1β; 1 ng/ml TGF-β) and time-dependent (IL-1β 12-hr peak; TGF-β 24-hr peak). Effect of IL-1β and TGF-β on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti-C1q antibody, and by binding studies with ¹²⁵l-C1q. Cells treated with cytokines displayed a two- to four-fold increased fluorescence of cell-bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL-1β (4.7 × 10⁶/cell) and TGF-β (3.9 × 10⁶/cell)-treated cells, compared to control (3.0 × 10⁶/cell), but had no effect on binding affinity. Rates of internalization of receptor-bound C1q were similar in cytokine-treated cells and controls. We propose from these data that IL-1β and TGF-β have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate-bound C1q. © 1993 Wiley-Liss, Inc.     

Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qR) has been produced by fusion of the P3 X 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 X 10(7) cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: this antibody competes for C1q binding sites on C1qR-bearing cells; the molecule recognized by this MAb is the C1qR; and cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125I-MAb detected a major protein band of approximately 85,000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125I-II1/D1 binding experiments revealed that the antibody bound to Raji cells or U937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant KD is 2.9 X 10(-10) M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 X 10(6) cells, giving an estimated 7.8 X 10(3) antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific and dose-dependent manner. These results indicate that the II1/D1 is a specific antibody directed against the C1q and can be a useful tool in studying the biologic interaction of human C1q with its receptors on a variety of cells.     

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.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites.

Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 X 10(-20) mol/cell per s at 25 degrees C compared with 254 X 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).     

Human blood platelets possess specific binding sites for C1q

Although platelet interactions with C1q are implied by the inhibitory effect of C1q on collagen-induced platelet aggregation, specific receptors have not as yet been identified. To address the question of platelet receptors for free C1q, direct radioligand binding studies were performed by using human blood platelets and purified, 125I-labeled C1q, and a monoclonal antibody (II1/D1) (IgM, lambda) directed against C1q receptors on peripheral blood leukocytes. Washed platelets bound both purified 125I-labeled C1q and II1/D1 in a specific and saturable manner under physiologic ionic strength conditions. At equilibrium, approximately 4000 molecules of C1q bound per platelet with an apparent dissociation constant of 3.5 X 10(-7) M. Maximum C1q binding was achieved in 5 min and correlated well with inhibition of collagen-induced platelet aggregation. Equilibrium binding of 125I-labeled II1/D1 to washed platelets required an incubation period of 15 to 30 min and II1/D1 concentrations approaching 50 micrograms/ml. Approximately 2000 molecules of II1/D1 bound per platelet, with an apparent dissociation constant of 2.8 X 10(-8) M. II1/D1 binding could be inhibited by the collagenous tail of C1q (c-C1q), suggesting that platelet receptors for these ligands are either the same or in close proximity. The data demonstrate that human blood platelets possess specific and saturable binding sites for free C1q that may function as collagen receptors, and may antigenically resemble C1q receptors on peripheral blood leukocytes.     

Binding of human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition

Purified human tumor necrosis factor (TNF) was iodinated to high specific activity with good retention of its biological activity, as determined by the cytotoxic titer on murine L929 cells. The binding of 125I-TNF to L929 and human HeLa S2 cells grown in monolayer was initially measured, but high levels of nonspecific binding were observed. Specific binding to high affinity receptors of HeLa S2 cells grown in suspension culture was demonstrated by competitive displacement experiments and analysis of the binding data with the LIGAND program. A KD of 2 X 10(-10) M and 6000 receptors/cell were calculated in this way. These observations provide the first direct evidence for a cellular receptor for TNF. The cell-bound 125I-TNF was internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and subsequently degraded to acid-soluble products. Three lines of human lymphoblastoid cells were examined for sensitivity to the cytostatic effect of TNF and for the presence of high affinity receptors. Daudi and Raji cells were insensitive to TNF and showed very few specific binding sites when incubated with 125I-TNF. Jurkat cells were growth-inhibited by TNF and showed a significantly greater number of specific binding sites than the other lymphoblastoid cells. These findings suggest that the sensitivity of some cell lines to the biological effects of TNF may be correlated with the presence of a relatively high number of receptors for this factor.     

Immunoglobulin M possesses two binding sites for complement subcomponent C1q, and soluble 1:1 and 2:1 complexes are formed in solution at reduced ionic strength

Soluble complexes were formed between C1q, a subunit of the first component of human complement, and four different Waldenstr?m IgM proteins at reduced ionic strengths. The equilibria between these complexes and the free proteins were studied in the ultracentrifuge. Complex formation was found to be a very sensitive function of the salt concentration, and at physiological ionic strength complex formation was negligible. The complexes were cross-linked with a water-soluble carbodiimide and separated by sucrose gradient centrifugation. Both 22 S 1:1 and 26 S 2:1 C1q X IgM complexes were formed; stoichiometry was established by cross-linking 125I-C1q with 131I-IgM and determining the ratios of the specific activities of the gradient-purified materials. The association process was studied as a function of protein concentration and was analyzed by Scatchard and Hill plots to yield stoichiometry, association constant, and degree of cooperativity. The results indicated that IgM has two identical and independent binding sites for C1q. The intrinsic association constant was found to vary between 10(6) M-1 at 0.084 M ionic strength to 10(4) M-1 at physiological ionic strength; the slope of the log-log plot gave a value of -6.0. The cross-linked complexes were examined by electron microscopy, and the C1q appeared to be attached to the IgM through the C1q heads, implying that the biologically significant binding sites were involved in this interaction. For the 2:1 complexes, the two C1q appeared to attach to opposite surfaces of the IgM, suggesting the presence of a pseudo-2-fold axis lying in the plane of the IgM disk.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.     

Diphtheria toxin receptor sites on membranes of cultured cells and erythrocytes demonstrated by fluorescence and electron microscopy

Diphtheria toxin (DT) receptor sites were identified on cell membranes by indirect immunofluorescence and immunoferritin labelling. The results obtained were correlated (1) with the binding of 125I-labelled DT (to the very same cell populations used in the ultrastructural studies), and (2) with toxin sensitivity as shown by inhibition of 14C-amino acid incorporation into protein. Binding and immunocytochemical studies were carried out at 0 degrees C to preclude internalization of DT. Receptor sites for DT were observed on functionally responsive, cultured human diploid (BUD-8) fibroblasts as well as on a small fraction of erythrocytes (RBC) of DT-susceptible species, i.e. human and sheep. On all BUD-8 cell sections DT receptor sites were located in annular patches and not on microvilli as had been observed in the KB epithelial cell line. Immunoferritin label was revealed on only 10% +/- 2.5 (SD) of human RBC in discreet loci along the cell membrane. Specificity of binding of DT to human fibroblast and RBC receptor sites was further demonstrated by competition between 125I-labeled and non-labelled DT. There was no specific binding to DT-treated cultured mouse L929 fibroblasts or to RBC of mouse of rat, species resistant to the toxin. Thus, direct ultrastructural immunocytochemical evidence is provided for the presence of specific cellular DT receptors on cultured cells and erythrocytes of species sensitive to DT, but not on those of species resistant to DT.     

Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.     

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with [3H]propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. [3H]hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of [3H]hTNF-beta to the cells. We conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.     

Modulation of class I and class II histocompatibility antigens on human T cell lines by IFN-gamma

IFN-gamma is an immunomodulatory agent which is known to induce or enhance the expression of class II histocompatibility Ag (Ia Ag) on many lymphoid cells and cell lines of diverse origin. However, we have observed that IFN-gamma did not induce the expression of Ia Ag on Ia- human T cell lines. Neither did IFN-gamma enhance the expression of Ia Ag on Ia+ T cells. However, IFN-gamma was able to enhance the expression of class I histocompatibility Ag (HLA-A,B,C Ag) on a number of the T cell lines tested. Experiments with 125I-labeled IFN-gamma showed a relatively small degree of specific binding to these T cell lines. More extensive studies on two of the T cell lines demonstrated 1000 and 2600 IFN-gamma binding receptor sites/cell and binding affinities of 4.0 X 10(-10) M and 7.3 X 10(-10) M. Thus, although IFN-gamma can bind to human T cell lines and enhance class I histocompatibility Ag on these cells, IFN-gamma alone does not appear to regulate expression of class II histocompatibility Ag on T cell lines.     

Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells

Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10(-10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein.     

OmpD but not OmpC is involved in adherence of Salmonella enterica serovar typhimurium to human cells

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 degrees C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.