Staphylococcal toxic shock toxin specifically binds to cultured human epithelial cells and is rapidly internalized.

Staphylococcal toxic shock syndrome toxin (TST) was labeled with 125I under mild conditions without apparent destruction of the molecule. [125I]TST bound specifically to human epithelial (Chang) cells in culture; the binding was inhibited by a 100-fold excess of unlabeled toxin. Scatchard analysis of the binding data indicated about 10(4) receptor sites per cell and a dissociation constant (Kd) of 4 X 10(-9) M. When cells pretreated with TST at 4 degrees C were swiftly transferred to 37 degrees C, the amount of surface-bound toxin rapidly declined, as determined by release of noninternalized label from the cell surface. Half-time (t1/2) of internalization was about 1.5 min. Ultrastructural studies showed that toxin labeled with ferritin-conjugated antibodies entered the cytoplasm via coated pits forming coated vesicles in the first 2 min of incubation at 37 degrees C. The coated vesicles coalesced with transport vesicles that are ultrastructurally unlike receptosomes. Thus, the unusual ultrastructural pattern of this internalization suggests that TST is initially internalized by receptor-mediated endocytosis and then enters an alternate pathway involving translocation in special transport vesicles, perhaps to other cells.     

Characteristics of histamine receptors present on suppressor T cells in "healthy individuals"

Twelve to 30% histamine receptor bearing cells were detectable in the peripheral blood mononuclear cells of healthy tuberculin sensitive individuals. The number of binding sites per cell ranged from 2.1 X 10(4) to 5.08 X 10(4) (mean 2.5 X 10(4)) with an affinity ranging from 2.5 X 10(-6) M to 10.9 X 10(-6) M (mean 3.6 X 10(-6) M). The histamine receptors on these cells were found to be of H2 type as indicated by the abrogation of binding of 3H-histamine by cimetidine. It was further confirmed that histamine receptor bearing cells in the peripheral blood belonged to a T cell subset which formed rosettes with AET treated sheep erythrocytes and had receptors for Fc portion of IgG and phenotype markers of T3 and T8. Deletion of such cells by means of affinity chromatography on histamine bound Sepharose columns, led to enhanced antigen induced lymphoproliferation indicating the suppressor nature of these T cells.     

Dermatophyte-hormone relationships: characterization of progesterone-binding specificity and growth inhibition in the genera Trichophyton and Microsporum

We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + and - mating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10(-7) to 2 X 10(-7) M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 X 10(-8) M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56 degrees C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding affinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings: DOC and DHT were effective competitors for [3H]progesterone binding. The growth of A. benhamiae + and -, M. canis, and T. rubrum were all inhibited by progesterone in a dose-responsive manner, with 50% inhibition achieved at concentrations of 9.8 x 10(-6), 1.2 x 10(-5), 1.5 x 10(-5), and 2.7 x 10(-6) M. respectively,.     

Immune responsiveness against the human placenta. II. Response with xenogeneic lymphocytes in culture.

Human placental plasma membrane vesicles were cultured for up to 5 days in the presence of spleen cells from (BALB/c X C57BL/6By) F1 hybrid mice. The membrane preparations either inhibited the uptake of [3H]-thymidine ([3H]-TdR) by destroying the viability of the T-cell population or stimulated weak lymphocyte division which was primarily an expansion of the T-cell population. This differential effect was dependent on the membrane concentration in culture and the length of time the membrane preparation had been stored. Membrane preparations that inhibited [3H]-TdR uptake could be converted into stimulatory-type membranes by preincubating them at 37 degrees for several days. This conversion coincided with a change in status concerning membrane susceptibility to disruption by cytotoxic non-T-cells present in the spleen of unimmunized animals. The conversion from stimulating-type membrane into inhibitory-type was never observed. Throughout these cultures the generation of cell-mediated cytolysis could not be detected.     

Solubilization and characterization of moderate and high affinity histamine binding sites on human blood mononuclear cells

The Triton X-100 solubilized extract of human peripheral blood mononuclear cells, in direct binding studies with 10(-9)-10(-6) M [3H]histamine contained both high and moderate affinity sites whose dissociation constants (Kd 4.4 X 10(-9) and 6.7 X 10(-7) M) were commensurate with basal plasma histamine levels and plasma levels obtained following physiological or mild pathological stimuli, respectively. Binding was enhanced by mM concns of calcium cations and by the protease inhibitor Pepstatin A. It was inhibited by bacitracin, agents interfering with thiol groups, Triton X-100 concns greater than 0.2% and EDTA. Binding was optimal between the pH range of 7.0 and 8.5 and was enriched for in a plasma membrane preparation. Thus the histamine binding sites identified maintained their specific ligand binding properties after solubilization from the cell surface and displayed properties fulfilling the criteria for receptors.     

Specific binding of radiolabeled membrane vesicles by T cells activated in the mixed lymphocyte reaction.

An assay was developed to quantitate binding of radiolabeled membrane vesicles by T cells activated in the primary mixed lymphocyte reaction (MLR). T blasts generated in unidirectional, reciprocal MLR combinations were found to bind much more effectively membrane vesicles prepared by nitrogen cavitation from allogeneic stimulator cells than from syngeneic spleen and lymph node cells which had been previously labeled biosynthetically with [³H]leucine. Specific inhibition of binding with cold vesicles prepared from stimulator cells but not from responder cells was observed. The number of specific antigen-binding cells is proportional to the concentration of membrane vesicles used. The proportion of labeled cells increases between 15 and 60 min of incubation with membrane vesicles, but thereafter remains constant. The binding of stimulator material is H-2-specific: cells bind membrane vesicles from congeneic mouse strains only if they share the H-2 antigens of the original stimulator strain. Responder blasts bind labeled stimulator membrane fragments after as early as 2 days of primary MLR culture; no binding of membrane vesicles by small lymphocytes was detected between 2 and 5 days of culture. However, between 8 and 12 days of culture, a significant proportion of small lymphocytes bind specifically stimulator membrane vesicles. The recognition function is sensitive to trypsin treatment but is regenerated within 5 to 6 h.     

Ciclosporin-Induced Immunosuppression in Vitro

The purpose of the present study was to analyse and correlate variations in lymphocyte sensitivity to, and binding of, ciclosporin (CsA) in vitro. Peripheral blood lymphocytes from healthy individuals were harvested over a 5-week period and activated with purified protein derivative (PPD) or alloantigens in the presence or absence of CsA (1 microgram/ml). Sensitivity to CsA was expressed as the ability of the drug to suppress cell proliferation ([3H]thymidine incorporation) and high-affinity interleukin-2 receptor (IL-2R) expression. Binding capacity was tested in a [3H]CsA binding assay. A significant variability in both sensitivity and binding capacity was recorded between individuals (P less than 0.001). There was no correlation between high sensitivity and high binding capacity. The intraindividual day-to-day variability did not differ significantly from the experimental (intra- and interassay) variability. The CsA-induced suppression of high-affinity IL-2R expression varied between 57.1 and 98.9%, while suppression of [3H]thymidine incorporation varied between 81.0 and 97.4%. Specific binding of 10 nM [3H]CsA at 37 degrees C varied between 5.4 and 10.7%.     

IL-2 responsiveness of lectin-induced lymphoblasts: soluble IL-2 receptor release and differential in vitro effects of immunosuppressants.

We examined the mode of action of different immunosuppressants on the responsiveness of phytohemagglutinin (PHA)-induced lymphoblasts further stimulated by recombinant interleukin-2 (rIL-2). The stimulation of PHA blasts with rIL-2 resulted in an enhancement of tritiated thymidine ([3H]TdR) incorporation and of soluble interleukin-2 receptor (sIL-2R) release. Cyclosporin A (CsA) and prednisolone inhibited in different ways the responsiveness of PHA pre-stimulated blood mononuclear cells (PBMC) to rIL-2, as measured by [3H]TdR incorporation. The addition of CsA resulted in considerable enhancement of the release of sIL-2R, whereas the addition of prednisolone was associated with a similar enhancement only when the higher concentrations of rIL-2 were employed. EGTA, a calcium (Ca2+) chelator, and verapamil, a Ca2+ channel blocker, inhibited [3H]TdR incorporation in a concentration-dependent manner. EGTA inhibited sIL-2R release in the same manner when used alone, and reversed the CsA- and prednisolone-induced enhancement of sIL-2R release by rIL-2 induced lymphoblasts, when used in combination with CsA or prednisolone. Verapamil had a similar but less striking effect. The effects of CsA and prednisolone were also studied in PHA-induced blasts originating from purified CD4+ or CD8+ lymphocytes. Stimulation of these blasts with rIL-2 resulted in higher [3H]TdR incorporation by CD8+ blasts than by CD4+ blasts: however, no sIL-2R release was detected in supernatants of either CD4+ or of CD8+ blasts. Both CsA and prednisolone inhibited the rIL-2-induced enhancement of [3H]TdR incorporation by both T-cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of 3H-histamine interaction with lymphocytes: receptor binding or uptake?

The immunoregulatory role of histamine is presumably mediated by specific receptors on the plasma membrane of lymphocytes. However, using murine spleen cells and a whole cell assay commonly applied in hormone receptor studies, specific histamine receptors with an affinity higher than that of non-specific binding could not be identified. Nevertheless, approximately 30% of the totally bound histamine was undissociable over a range of added histamine concentrations (9 X 10(-6)-1 X 10(-2) M). Lectin stimulation of spleen cells caused an additional two-fold increase of undissociable histamine. The H1 receptor antagonist, diphenhydramine, blocked histamine uptake, whereas the H2 receptor antagonist, cimetidine, had no effect. Binding experiments carried out at 4 degrees C demonstrated that the amount of undissociable histamine was much reduced. Even at 4 degrees C, evidence for specific membrane associated histamine receptor could not be obtained. It was therefore concluded that lymphocytes take up histamine by an energy-dependent mechanism inhibitable by diphenhydramine but not cimetidine, and that the usual hormone receptor methodology did not allow the identification of specific membrane associated histamine receptors.     

Differences in Binding Sites for Prostaglandin E2 on Mononuclear Leucocytes from Human Newborns and from Their Mothers

Binding of [3H]prostaglandin E2 (PGE2) to peripheral mononuclear leucocytes (PML) from newborns and their mothers was studied. Specific binding of PGE2 to both maternal and neonatal PML was found. The binding was maximal after 15 min of incubation at 37 degrees C and specific for PGE1 and PGE2 versus PGA1, PGF1 alpha, and PGF2 alpha. The amount of PGE2 specifically bound to neonatal PML was about 30% of the amount bound to maternal PML. The average number of binding sites for PGE2 on maternal PML was calculated to 1800 per cell, and the dissociation constant (KD) was 6.5 X 10(-9)M. The corresponding figures for neonatal PML could not be calculated owing to the low number of binding sites on those cells. After preincubation for 18 h the binding of PGE2 to maternal PML was decreased and equalled that of PGE2 to neonatal PML, whereas neonatal PML were unaffected by preincubation. These results indicate that differences in sensitivity to suppression by PGE and the effect of preincubation may be linked to differences in binding of PGE.     

Aldosterone binding in isolated tubules I. Biochemical determination in proximal and distal parts of the rabbit nephron

Microbiochemical methods were applied to proximal tubules (PCT) and a mixture of distal and cortical collecting tubules (D + C) of rabbit kidney in order to define aldosterone binding sites. For each experiment, after incubation of kidney pyramids with [3H]aldosterone ([3H]A), either alone or in the presence of an excess unlabeled A, 100-150 mm of both categories of tubules were microdissected using collagenase. Specific binding was determined on the nuclear fraction of each sample. Aldosterone concentrations ranged from 2 X 10(-9) to 4.5 X 10(-8) M. No specific binding was detectable in PCT. Specific binding in D + C increased rapidly as a function of [3H]A concentration up to 5 X 10(-9) M and then more slowly. No plateau was reached. Both the absence of saturation of the binding curve and the curvilinear aspect of the Scatchard plot suggested the presence of two binding sites, one of high affinity, presumably a mineralocorticoid site, and the other of lower affinity, possibly a glucocorticoid site. These experiments suggest that the distal structures of the nephron, located in the cortex, are the main sites of binding of aldosterone and contain a high number of specific binding sites for this hormone.     

Localization of [3H]ouabain-sensitive Na+ pump sites in cultured pig kidney cells.

Pig kidney cells, LLC-PK1, grown by standard tissue-culture techniques form monolayers and maintain morphological features characteristic of epithelia. Cultures exposed to 2 X 10(-6) M [3H]ouabain for 30 min at 37 degrees C bound 7.77 +/- 0.37 pmol/mg protein. This could be reduced by 58% by incubation in the presence of 45 mM K+. Freeze-dry radioautographic localization of [3H]ouabain-binding sites revealed grains distributed only along that fraction of the plasmalemma directly facing the culture-dish surface. Binding and localization of [3H]ouabain were correlated with an inhibition of the Na+ pump in these cells because analysis of cellular electrolytes in control cultures versus those exposed to 10(-3) M ouabain revealed a fall in K+ from 419 +/- 9 to 173 +/- 4 mmol/kg dry wt with a reciprocal increase in Na+. There was no change in cell H2O. Similarly, oxygen consumption was reduced by 32% after exposure to ouabain. These results provide direct evidence that in epithelial cells in culture the membrane facing the culture dish corresponds to the basolateral membrane of epithelial cells in vivo.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils: change of receptor affinity and number by L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)

Pretreatment of rabbit peritoneal neutrophils at 37 degrees with 10-35 microM L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) decreases by 20-50% the detectable number of f Met-Leu-[3H]Phe binding sites. Greater TPCK concentrations, between 50 and 100 microM, cause less of a decrease or actually increase peptide binding activity to a level greater than that of untreated cells. Furthermore, Scatchard analysis indicates that the sites detected on neutrophils after TPCK treatment have 1.2-3.2 fold lower apparent Kd (higher affinity) than those detected on untreated, control cells (1.1 +/- 1.7 X 10(-8) M vs 1.7 +/- 1.5 X 10(-8) M, P less than 0.02). Thus, TPCK treatment of rabbit peritoneal neutrophils causes both a decrease in f Met-Leu-[3H]Phe receptors and increases the affinity of the remaining sites. In addition, peritoneal neutrophils incubated at 37 degrees without TPCK were found to rapidly express additional f Met-Leu-[3H]Phe receptors. These additional sites, however, were not evident on neutrophils incubated at 37 degrees with TPCK. Concomitantly with the expression of additional sites, neutrophils placed at 37 degrees were found to spontaneously release small amounts of lysozyme. However, since equivalent amounts of lysozyme were released by cells incubated with or without TPCK, we are unable to state whether expression of the additional sites is due to neutrophil degranulation. Finally, although rabbit peripheral blood neutrophils also show an increase in binding sites at 37 degrees, treatment of these cells with TPCK does not cause a decrease in their f Met-Leu-[3H]Phe binding activity.     

Inhibition of interleukin 4 receptor expression on human lymphoid cells by cyclosporin

The effect of the immunosuppressant cyclosporin (CsA) on the expression of interleukin (IL) 4 membrane receptors on human peripheral blood mononuclear cells (PBMC) was investigated after cell activation by anti-CD3 antibody, IL 2 or IL 4. Previous studies with 125I-labeled IL 4 identified on resting lymphocytes a trimolecular complex consisting of a 65/70-kDa doublet and a 110-kDa protein with approximately 300 high-affinity binding sites (Kd 100 pM) and approximately 9000 low-affinity binding sites (Kd 30 nM). Upon cell activation by anti-CD3 antibody both low- and high-affinity binding sites increased about threefold concomitant with up-regulation of all the cross-linked proteins. CsA inhibited anti-CD3 antibody-induced up-regulation of IL 4 receptor (IL 4R)-associated proteins as well as the expression of high-affinity binding sites. However, up-regulation of IL 4R by its own ligand or IL 2 and the growth-promoting effect of IL 4 on activated, IL 4R+ T cells were CsA resistant. Since CsA inhibits the synthesis of IL 4, exogenous IL 4 was added to the cultures and it partially reversed the inhibitory effect of CsA on cell proliferation as well as on IL 4R expression. It is concluded that the inhibitory effect of CsA on IL 4R expression may contribute to the immunosuppressive effect of the drug.     

Bacterial aggregating activity in human saliva: simultaneous determination of free and bound cells.

Two new assays for saliva-mediated aggregation of oral bacteria have been developed, based on the use of [3H]thymidine-labeled cells. One assay separates free cells from aggregated cells by centrifugation through sucrose, whereas the other utilizes membrane filters (8 micrometers, Nuclepore) to effect the separation. Comparison of these assays with the turbidity method reveals that they are faster (X20 to 40) and require 10 times less saliva and bacteria. The aggregation of Streptococcus sanguis M5, as determined with these assays, is complete in 5 min and is dose dependent on added cells and saliva. The reaction exhibits a temperature optimum of 42 degrees C with no reaction at 0 degrees C. If the pH is reduced to below 5, saliva-dependent aggregation is inhibited. The salivary factor(s) are heat labile, losing 100% of their activity after 100 degrees C, 10 min or 70 degrees C, 30 min.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells.

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Ligand-Induced Redistribution and Augmentation of Surface-Bound Myeloma Protein on MOPC315 Plasmacytoma Cells

The dinitrophenyl (DNP)- and trinitrophenyl (TNP)-binding IgA(lambda2) myeloma protein M315, bound on the surface of MOPC315 mouse plasmacytoma cells, was redistributed into spots, patches, and, more rarely, into caps by TNP14-BSA and by divalent but not monovalent anti-M315 antibodies. Antiserum to the L-chain of M315 (L315) induced similar redistribution of L315 bound on the surface of variant cells that only produced L315. The spots were much larger and more brilliant when the cells were incubated with the ligands at 37 degrees C than at 4 degrees C. Redistribution of M315 also occurred on M315-producing cells in peritoneal diffusion chambers incubated in BALB/c mice producing antibodies against the M315 idiotype. The clearance of immune aggregates and the regeneration of new surface-bound M315 in diffusion chambers were much slower for MOPC315 cells than that reported for B lymphocytes. The total pool of M315 was 1.9 pg per cell (about 8 X 10(6) 7S molecules), but only an average of 6 X 10(3) [125I]TNP-BSA molecules were bound on the surface of each MOPC cell at 4 degrees C. The amount of surface-bound TNP-BSA increased eightfold when the cells were preincubated at 37 degrees C with rabbit anti-mouse IgA; at 4 degrees C the increase was only twofold. The data indicate that multivalent ligands specific for M315 induce an accumulation of M315 on the cell surface that correlates with secretion; the immediate precursors of secreted myeloma protein may be arrested in their transit through the membrane by the ligands.     

Assessment of the antidepressant activity of dothiepin and its metabolites by preclinical tests

The affinities of dothiepin and its principal metabolites northiaden, dothiepin sulphoxide and northiaden sulphoxide for [3H]imipramine binding sites in the rat cortical homogenates, and for [3H]spiperone and [3H]serotonin receptor sites in preparations from the rat frontal cortex and hippocampus were studied. As inhibitors of [3H]imipramine binding, the strengths of the drugs are, in terms of their IC50 (concentration corresponding to 50% inhibition): dothiepin 2.8 X 10(-6) M, northiaden 5.0 X 10(-6) M, northiaden sulphoxide 4.0 X 10(-5) M and dothiepin sulphoxide 3.2 X 10(-5) M. The potencies of the drugs in inhibiting serotonergic binding followed a similar trend. Using frontal cortical tissue suspensions and [3H]spiperone, the IC50 values were determined to be: dothiepin 4.2 X 10(-6) M, northiaden 5.0 X 10(-6) M, northiaden sulphoxide 1.6 X 10(-4) M and dothiepin sulphoxide 1.6 X 10(-4) M; whereas in hippocampal suspensions and using [3H]serotonin, the IC50 values were 2.5 X 10(-6) M, northiaden 4.0 X 10(-5) M, dothiepin sulphoxide 2.5 X 10(-4) M and northiaden sulphoxide greater than 10(-3) M. The influence of the drugs on the uptake of [14C]serotonin into human platelets was also investigated. All had an inhibitory effect upon the uptake, the order of potency being dothiepin greater than northiaden greater than northiaden sulphoxide greater than dothiepin sulphoxide. Plots of 1/v versus 1/s showed that the inhibition was competitive for all four compounds.