IL-3 up-regulates and activates human eosinophil CD32 and αMß2 integrin causing degranulation.

BACKGROUND: Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. OBJECTIVE: We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. RESULTS: Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation. When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. CONCLUSIONS AND CLINICAL RELEVANCE: We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.

IL-5-stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8-dependent migration.

BACKGROUND: IL-5 causes suspended eosinophils to polarize with filamentous (F)-actin and granules at one pole and the nucleus in a specialized uropod, the "nucleopod," which is capped with P-selectin glycoprotein ligand-1 (PSGL-1). IL-5 enhances eosinophil adhesion and migration on periostin, an extracellular matrix protein upregulated in asthma by type 2 immunity mediators. OBJECTIVE: Determine how the polarized morphology evolves to foster migration of IL-5-stimulated eosinophils on a surface coated with periostin. METHODS: Blood eosinophils adhering to adsorbed periostin were imaged at different time points by fluorescent microscopy, and migration of eosinophils on periostin was assayed. RESULTS: After 10 minutes in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30-60 minutes, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. CONCLUSIONS AND CLINICAL RELEVANCE: Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target.

Anti-IL-5 attenuates activation and surface density of ß(2) -integrins on circulating eosinophils after segmental antigen challenge.

BACKGROUND: IL-5 activates α(M) ß(2) integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated ß(2) -integrins. OBJECTIVE: To identify roles for IL-5 in regulating human eosinophil integrins in vivo. METHODS: Blood and BAL eosinophils were analysed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. RESULTS: Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated ß(2) -integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil ß(2) , α(M) and α(L) integrin subunits increased modestly post challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of ß(2) , α(M) , α(L) and α(D) and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity α(M) ß(2) , were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of ß(1) -integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of ß(1) -integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared with eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. CONCLUSION AND CLINICAL RELEVANCE: IL-5 supports a heterogeneous population of circulating eosinophils with partially activated ß(2) -integrins and is responsible for up-regulation of ß(2) -integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has minimal effects on properties of eosinophils in BAL. Dampening of ß(2) -integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5.

Assembly of extracellular matrix.

A great challenge in understanding how different extracellular matrices assemble is to sort through the vast number of possible interactions between and among matrix molecules. The most profound insights are likely to come from patients with defined defects of matrix molecules and the use of transgenic mice or other experimental technologies that mimic the complexity of the human system.

Synthesis of fibronectin by cultured human endothelial cells.

Plasma fibronectin is probably the major nonimmune particulate opsonin in blood and is cross-linked to fibrin during the final stage of blood coagulation. Fibronectin also occurs in an insoluble form in basement membranes especially those underlying endothelial cells and in loose connective tissue. Fibronectin was demonstrated in cultured human endothelial cells and in the surrounding extracellular matrix by immunofluorescence microscopy by using antibody to human plasma fibronectin. Cultured human endothelial cells released fibronectin into the culture medium which was immunologically identical to the fibronectin in human plasma. Cultured human endothelial cells were labeled with [3H] leucine. The radioactive fibronectin present in the endothelial postculture medium and in urea extracts of cellular monolayers was isolated with either anti-fibronectin coupled to Protein A-Sepharose or double antibody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When reduced, the [3H] fibronectin synthesized by cultured endothelial cells had the same mol wt (approximately 200,000) as plasma fibronectin. Unreduced, the [3H] fibronectin synthesized by endothelial cells migrated as a dimer, as did plasma fibronectin. Fibronectin accounted for approximately 15% of the protein synthesized and released by endothelial cells into the culture medium. Thus, cultured endothelial cells synthesize fibronectin, secrete it into the culture medium, and incorporate it into extracellular matrix. The results suggest that the endothelial cell is potentially a major site of synthesis of circulating plasma fibronectin. In addition, fibronectin derived from endothelial cells may be an important structural component of the subendothelium.

Synthesis and secretion of alpha2-macroglobulin by cultured human fibroblasts.

The following observations indicate that cultured human WI-38 fibroblasts synthesize and secrete alpha2-macroglobulin into serum-free medium: (a) after incubation of cultures with [35S]L-methionine, a labeled protein appeared in the medium which was precipitated by antiserum directed against alpha2-macroglobulin; (b) after incubation of cultures with [35S]L-methionine, a major band of radioactivity detected by polyacrylamide gel electrophoresis of the proteins in medium co-migrated with alpha2-macroglobulin; and (c) the amount of alpha2-macroblobulin in the medium, estimated both functionally and immunologically, increased with time in normal but not not puromycin-treated cultures.

Localization of cell surface sites involved in fibronectin fibrillogenesis.

Fibronectin binding sites on cultured human fibroblasts were localized by high voltage electron microscopy using either 5- or 18-nm colloidal gold beads (Au5 or Au18) bound to intact fibronectin, the 70-kD amino-terminal fragment of fibronectin that blocks incorporation of exogenous fibronectin into extracellular matrix, or 160-180-kD fragments of fibronectin with cell adhesion and heparin-binding activities. Binding sites for Au18-fibronectin on the cell surface were localized to specific regions along the edge of the fibroblast and on retraction fibers. Au18-fibronectin complexes at these sites were initially localized in clusters that co-aligned with intracellular microfilament bundles. With longer incubations, Au18-fibronectin complexes were arranged into long fibrillar networks on the cell surface and in the extracellular space. The appearance of Au18-fibronectin in these fibrillar networks and disappearance of clusters of Au18-fibronectin suggest that Au18-fibronectin complexes are arranged into matrix at specific regions of the cell surface. Au18-70-kD fragment complexes initially had a similar distribution to Au18-fibronectin complexes. With longer incubations, Au18-70-kD fragment complexes were found in long linear arrangements on the cell surface. Double labeling experiments using Au18-70-kD fragment and Au5-160-180-kD fragments showed that the 70-kD fragment and the 160-180-kD fragments bind to different regions of the cell.

Interactions of thrombospondin with endothelial cells: receptor-mediated binding and degradation.

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.

Disulfides modulate RGD-inhibitable cell adhesive activity of thrombospondin.

Thrombospondin (TSP) contains the Arg-Gly-Asp (RGD) sequence that is thought to be important for cell adhesion mediated by several cell-surface integrin receptors. The RGD sequence is located in the type 3 repeat region of TSP that has multiple Ca2+ binding sites and is subject to a complex intramolecular thiol-disulfide isomerization. TSP that we isolated from thrombin-activated human platelets using buffers containing 0.1 mM Ca2+, in which Cys974 is the major labeled cysteine, did not have RGD-inhibitable adhesive activity. However, one of our preparations of TSP and TSP purified following alternative procedures using greater than or equal to 0.3 mM Ca2+ did have RGD-inhibitable adhesive activity. Reduction of TSP with DTT, either before or after adsorption to surfaces, enhanced its adhesive activity. Reduced TSP supported robust cell spreading when coated at concentrations as low as 1 micrograms/ml, whereas "adhesive" TSP not treated with DTT was active at coating concentration of greater than 20 micrograms/ml and supported only modest cell spreading. Lower DTT concentrations were required for enhancement of the adhesive activity of TSP if Ca2+ was chelated with EDTA. Cellular adhesion to DTT-treated TSP was inhibited by RGD-containing peptide and by mAb to a functional site of the alpha v beta 3 integrin. Cell blots of reduced proteolytic fragments of TSP localized the adhesive activity to the RGD-containing type 3 repeat region. These results suggest a novel mechanism for regulation of integrin-ligand interactions in which the ligand can isomerize between inactive and active forms.

Binding of plasma fibronectin to cell layers of human skin fibroblasts.

Human plasma fibronectin bound to confluent cell layers of cultured human-skin fibroblasts in two distinct pools. Initial binding of fibronectin occurred in a deoxycholate-soluble pool (Pool I). Binding in Pool I was reversible and reached a steady state after 3 h. After longer periods of incubation, fibronectin became bound in a deoxycholate-insoluble pool (Pool II). Binding in Pool II was irreversible and proceeded at a linear rate for 30 h. After 30 h of incubation, a significant proportion of fibronectin bound in Pool II was present as disulfide-bonded multimers. HT1080 cells, a human sarcoma cell line, did not bind fibronectin in either pool. Also, isolated cell matrices prepared by deoxycholate extraction did not bind fibronection. Binding of fibronectin in Pool I of normal fibroblasts occurred via specific, saturable receptors. There were 128,000 binding sites per cell, and KDiss was 3.6 X 10(-8) M. Fluorescence microscopic localization of fibronectin bound in Pool I and Pool II was performed using fluorescein-conjugated fibronectin. Fluorescent staining in Pool I was present in a punctate pattern and in short, fine fibrils. Pool II fluorescence was exclusively in coarse, dense fibrils. These data indicate that plasma fibronectin may become incorporated into the tissue extracellular matrix via specific cell-surface receptors.

Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts.

Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P.J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.

Binding and degradation of platelet thrombospondin by cultured fibroblasts.

Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detected in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37 degrees C were 6 X 10(-8) M and 1.4 X 10(5) molecules per cell per minute, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4 degrees C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed two fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I-thrombospondin. The second fraction was localized to a trypsin-sensitive, fibrillar, extracellular matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I-thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations greater than 3 X 10(-8) M. The ability of 125I-thrombospondin to bind to extracellular matrix was diminished tenfold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. These results suggest that thrombospondin may play a transitory role in matrix formation and/or organization and that specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.

Synthesis and secretion of thrombospondin by cultured human endothelial cells.

Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine-labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin.

Modulation of beta1A integrin functions by tyrosine residues in the beta1 cytoplasmic domain.

beta1A integrin subunits with point mutations of the cytoplasmic domain were expressed in fibroblasts derived from beta1-null stem cells. beta1A in which one or both of the tyrosines of the two NPXY motifs (Y783, Y795) were changed to phenylalanines formed active alpha5 beta1 and alpha6 beta1 integrins that mediated cell adhesion and supported assembly of fibronectin. Mutation of the proline in either motif (P781, P793) to an alanine or of a threonine in the inter-motif sequence (T788) to a proline resulted in poorly expressed, inactive beta1A. Y783,795F cells developed numerous fine focal contacts and exhibited motility on a surface. When compared with cells expressing wild-type beta1A or beta1A with the D759A activating mutation of a conserved membrane-proximal aspartate, Y783, 795F cells had impaired ability to transverse filters in chemotaxis assays. Analysis of cells expressing beta1A with single Tyr to Phe substitutions indicated that both Y783 and Y795 are important for directed migration. Actin-containing microfilaments of Y783,795F cells were shorter and more peripheral than microfilaments of cells expressing wild-type beta1A. These results indicate that change of the phenol side chains in the NPXY motifs to phenyl groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility.

Inhibition of cell adhesion by high molecular weight kininogen.

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid.

Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet-poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino-terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.

Inhibition of binding of fibronectin to matrix assembly sites by anti-integrin (alpha 5 beta 1) antibodies.

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.

Co-assembly of plasma and cellular fibronectins into fibrils in human fibroblast cultures.

Exogenous plasma and endogenous cellular fibronectins on the surface of cultured fibroblasts and in extracellular matrix fibrils were colocalized by fluorescent and high voltage immunoelectron microscopy. Fibroblast cultures grown in the presence or absence of cycloheximide were incubated with exogenous plasma fibronectin labeled with fluorescein isothiocyanate. A monoclonal antibody specific for the EIIIA sequence of cellular fibronectin was used to detect cellular fibronectin. A rabbit antifluorescein antibody identified fluoresceinated plasma fibronectin. In cultures incubated in the presence of cycloheximide, plasma fibronectin was bound to the cell surface and was assembled into extracellular fibrils. In cultures grown in the absence of cycloheximide, plasma and cellular fibronectins were observed in the same matrix fibrils and in the same locations on the cell surface. There was not, however, random admixture of the two proteins.

Binding and factor XIIIa-mediated cross-linking of a 27-kilodalton fragment of fibronectin to Staphylococcus aureus.

A 27-kilodalton tryptic fragment, derived from the amino terminus of the 200-kilodalton fibronectin subunit, inhibited binding of intact fibronectin to Staphylococcus aureus and could be cross-linked to Staphylococcus aureus by blood coagulation Factor XIIIa. Interactions of fibronectin with Staphylococcus aureus via this fragment may be important for bacterial opsonization and attachment.

Ca2(+)-sensitive binding of thrombospondin to U937 cells is due to the formation of calcium precipitate in the binding medium.

Thrombospondin (TSP) binds to U937 monocytic cells in a Ca2(+)-enhancible and EDTA-inhibitable manner (Silverstein, R. L., and R. L. Nachman. 1987. J. Clin. Invest. 79:867-874; Silverstein, R. L., A. S. Asch, and R. L. Nachman. 1989. J. Clin. Invest. 84:546-552). We reproduced the results when RPMI cell culture medium, but not when HBSS was used as binding medium. Addition of 1 mM Ca2+ to RPMI medium increased the binding of TSP to suspended U937 cells more than eightfold; the increase was blocked by EDTA but not by heparin. Further studies showed that addition of 1 mM Ca2+ to RPMI medium resulted in an insoluble precipitate, which did not form when EDTA was present or when 1 mM extra Ca2+ was added to HBSS. TSP bound to the precipitate in a saturable and specific manner. The precipitate enhanced binding of TSP to MG63 osteosarcoma cells in a monolayer binding assay. Enhancement of binding in the monolayer assay was observed for fibronectin and vitronectin as well. Our data indicate that there is not a specific Ca2(+)-dependent TSP receptor on U937 cell surface. Instead, the extra binding enhanced by Ca2+ is due to the formation of insoluble salts in the medium.