Plasma Fn Research Articles

Characterization of a Novel Human Antibody Xenoreactive with Fibronectin

Recent reports have used bovine fibronectin (Fn) as source of antigen to study human anti-Fn autoantibodies. We have characterized a novel human antibody (Ab) reactive with bovine and marsupial Fn, but not with human Fn. Indirect immunofluorescence, wet cleaving and protein adherence assays, immunoblotting, blot-affinity purification, a cell adhesion inhibition assay, and competitive experiments with synthetic peptides were used to characterize the anti-Fn Ab in serum from a patient with an undifferentiated connective tissue disease. A characteristic Fn-like network was observed by indirect immunofluorescence on bovine MDBK and marsupial PtK 2 cells, but not on various human cell lines. Double immunofluorescence revealed colocalization of the Ab with a mouse monoclonal anti-Fn Ab. A reactive polypeptide of 240 kDa corresponding to the M r of Fn was identified by immunoblotting using MDBK and PtK 2 total cell lysates. The Ab reacted with the 240-kDa band of purified bovine Fn with an endpoint titer of 1:64,000, while no reactivity was observed with human cellular or plasma Fn. Blot-affinity purification of the Ab from the 240-kDa PtK 2 region confirmed that the Fn-like fluorescent pattern observed was due to reactivity with the 240-kDa band and not with other regions of the blot. The Ab affinity-purified from the 240-kDa region also reacted with purified bovine Fn by immunoblotting. Functional analysis disclosed specific inhibition of PtK 2 and MDBK cell adhesion by the affinity-purified anti-Fn Ab. Competitive experiments with synthetic peptides demonstrated that the epitope is located in the decapeptide RGDSPASSKP containing the cell-binding domain of Fn. Longitudinal analysis of the Ab revealed its persistence over 6 years. Bovine and marsupial Fn can be the focus of a highly specific and persistent human immune response. Reactivity of a human Ab with bovine Fn does not imply cross-reactivity with human Fn. In light of recent reports using bovine Fn to characterize human anti-Fn "autoantibodies," future studies on human anti-Fn should specifically employ purified human Fn as antigen.

Release of biological activities from quiescent fibronectin by a conformational change and limited proteolysis by matrix metalloproteinases.

We reported that specific biological activities are confined to three domains of the fibronectin (Fn) molecule [Fukai et al. (1991) J. Biol. Chem. 266, 8807; Fukai et al. (1993) Biochemistry 32, 5746]: the potent ability to stimulate the adipocyte differentiation of ST-13 cells is in the amino-terminal fibrin-binding (Fib 1) domain (referred to as Fib 1 domain activity); the RGD-dependent activities that stimulate NIH-L13 cell migration and inhibit adipocyte differentiation are in the central cell-binding (Cell) domain (Cell domain activity); and the activity that stimulates cell migration in a RGD-independent manner is in the carboxyl-terminal fibrin-binding (Fib 2) domain (Fib 2 domain activity). Human plasma Fn which was purified without exposure to a denaturant, such as urea, exhibited no Fib 1, Fib 2, or Cell domain activity. By exposure to urea or surface adsorption, Fn showed Cell domain activity but not those of the Fib 1 and Fib 2 domains. Whether the cryptic domain activities are disclosed or not depended on whether or not the responsible domains were irreversibly exposed from confined environments of Fn structure as confirmed by light-scattering measurement and enzyme immunoassay using domain-specific monoclonal antibodies. We then investigated the action of matrix metalloproteinases (MMPs) in liberating the Fib 1, Cell, and Fib 2 domain activities. Matrilysin released only the Cell domain activity. In contrast, stromelysin, collagenase, and especially gelatinase A additionally liberated the Fib 1 and Fib 2 domain activities. The Fib 1, Fib 2, and Cell domains acquired much higher activities when they were freed from linkage with adjacent domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibronectin and 130-kDa Molecule Complex Mimics Snake Venom Botrocetin-Like Structure Potentially Modulating Association between von Willebrand Factor and Vascular Vessel Wall

We established seven hybridomas secreting monoclonal antibodies (MoAbs) against the venom from Bothrops jararaca. Six of them were demonstrated to specifically recognize botrocetin, a venom protein which binds with von Willebrand factor (vWf) and induces platelet agglutination. Two of them, BCT4-3 and BCT115-2 MoAbs, could significantly inhibit botrocetin binding with plasma vWf. BCT4-3 could react slightly with a monolayer of human endothelial cells (ECs), and BCT4-3 binding to ECs was drastically enhanced by the coexistence of human plasma in a dose-dependent manner, indicating that a biological modulator structurally resembling botrocetin is created initially on the EC surface complexed with some plasma proteins. Botrocetin-like components could be immuno-purified only by immobilized BCT4-3, but not by the other immobilized MoAbs, from umbilical vein extracts. Interestingly, the immunoisolated materials were identified to consist essentially of fibronectin (Fn) and a 130 kDa molecule, and this complex bound to vWf in the extracts. Depletion of Fn from plasma decreased BCT4-3 binding to ECs. The epitope of BCT4-3 expressed on the endothelial surface, comprising plasma Fn and the coisolated 130 kDa molecule, is proposed to be a physiological modulator structurally mimicking botrocetin, and essentially supporting vWf-binding to injured endothelium and subsequently to circulating platelets.

Delayed elevation of ED1-cellular fibronectin in plasma following postsurgical bacteremia.

Fibronectin (Fn) exists in both a soluble form in plasma and lymph as well as an insoluble form in the extracellular matrix. Matrix-localized cellular fibronectin (cFn) contains extra domains (ED1 and/or ED2) not found in plasma Fn (pFn). Very little (< 1-2%) ED1-containing cFn exists in normal blood, and its rapid release into plasma and/or lymph is believed to reflect acute vascular injury. We used a polyclonal antibody to sheep pFn and a monoclonal antibody to ED1 domain of cFn to measure both pFn and ED1-cFn in relationship to lung lymph flow (QL), lung lymph-to-plasma (L/P) total protein concentration ratio, and lung protein clearance (LPC). Unanesthetized sheep (n = 7) were injected intravenously with Pseudomonas aeruginosa (5 x 10(8)) at both 2 and 7 days following surgical preparation of a lung lymph fistula. After both bacterial challenges, we observed an early increase in QL and a small decline in the L/P ratio (0-2 h), reflecting increased fluid filtration in the presence of an intact vascular barrier. This was followed by a further increase (P < 0.05) in QL; an elevation in the L/P ratio; and a marked (P < 0.05) increase in LPC over 3-6 h, indicative of an increase in lung endothelial protein permeability. Before the first bacterial infusion, ED1-cFn in plasma was 9.97 micrograms/ml or approximately 2% of the total Fn antigen in plasma; whereas ED1-cFn in lung lymph was 6-8% of total lymph Fn.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of ED1 fibronectin from matrix of perfused lungs after vascular injury is independent of protein synthesis.

Fibronectin (Fn) is an adhesive protein found in the plasma and extracellular tissue matrix. Locally synthesized tissue or cellular Fn (cFn) has extra domains (ED1 and ED2) not present in liver synthesized plasma Fn (pFn). In the lung, Fn is found in the endothelial and epithelial basement membranes, as well as in the interstitial matrix. Utilizing murine monoclonal antibodies to ED1 of cFn, we studied the release of total Fn as well as ED1-Fn into the plasma-free perfusate of the isolated perfused rabbit lung in relation to changes in lung weight due to fluid accumulation after oxidant (H2O2) challenge. Both parameters were also studied after addition of cycloheximide (20 micrograms/ml perfusate) to the perfusion medium to inhibit lung protein synthesis. After continuous H2O2 challenge (11 nmol.ml buffer-1.min-1), there was a 2.25 +/- 0.62 g increase in lung weight over 60 min. Measurement of 125I-labeled albumin clearance at 20 min after the start of H2O2 infusion confirmed an increase in lung endothelial protein permeability after H2O2 treatment. Fn antigen was released into the perfusate as early as 15 min after oxidant challenge. By 60 min, total perfusate Fn increased in H2O2-treated lungs (n = 6) to 2.10 +/- 0.48 micrograms/ml compared with only 0.35 +/- 0.09 micrograms/ml in normal control lungs (n = 5). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of nonreduced samples revealed that the Fn released consisted of primarily intact (440 kDa) Fn as well as Fn fragments. A rapid release of ED1-Fn paralleled the increased release of total Fn.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C modulation of fibronectin matrix assembly.

Fibroblasts have cell surface sites that mediate the assembly of fibronectin (Fn) into the extracellular matrix. Treatment of fibroblasts with kinase inhibitors (ML-7, H7, HA1004, calphostin C, and staurosporine) resulted in the rapid decrease in the binding of 125I-labeled plasma Fn and iodinated amino-terminal fragments of Fn. The dose responses of the four inhibitors suggest that the target kinase is protein kinase C (PKC) rather than the cyclic AMP- or cyclic GMP-dependent kinases. Three different fibroblastic cells were similarly affected. The inhibition was rapid and reversible and could not be overcome by increasing concentrations of Fn. Treatment of fibroblasts with phorbol esters and other agents that activate PKC resulted in increased amounts of 125I-labeled Fn binding to the cell surface. These results imply that Fn matrix assembly is modulated by PKC-mediated phosphorylation.

ED1-containing cellular fibronectin release into lung lymph during lung vascular injury with postoperative bacteremia.

Fibronectin (Fn) exists in both a soluble and insoluble form. Soluble Fn in plasma and lymph is an opsonic molecule that enhances phagocytic host defense. Insoluble Fn in the subendothelial and extracellular matrix is an adhesive molecule that mediates cell adhesion to substratum. The extracellular matrix of tissues such as the lung contains a mixture of both plasma-derived fibronectin (pFn) as well as locally synthesized cellular fibronectin (cFn). cFn is antigenically related to pFn, but cFn has extra domains (ED1 and ED2) that do not exist in liver synthesized pFn. The purpose of this study was to determine whether ED1-Fn was released into lung lymph before an increase in lung vascular permeability following postoperative bacteremia. Male sheep (n = 8) with surgically prepared lung lymph fistulae were infused intravenously with a sublethal dose (5 x 10(8)) of Pseudomonas aeruginosa 2 days following surgery. Lymph flow (QL), lymph-to-plasma (L/P) total protein ratio, lung protein clearance (QL x L/P), and hemodynamics were measured over 48 h following bacterial challenge. The lymph and plasma ED1-Fn concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using a murine monoclonal antibody specific to the ED1 region of human cFn. There was a rapid rise of ED1-Fn flux in lung lymph which was evident 60 min after the start of bacterial infusion, resulting in a maximum three- to fourfold increase (P < 0.05) in this parameter. In contrast, the ED1-Fn concentration in plasma before bacterial infusion was less than lung lymph and it did not increase over the initial 6 h following bacterial infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Fibronectin in Immune Glomerulonephritis

There is already a considerable amount of evidence suggesting that fibronectin (Fn) plays an important role in the pathogenic process in some forms of glomerulonephritis (GN). It has been postulated that Fn may participate in the progression or regression of glomerular diseases. The Fn is presented in the kidney as a normal component of the mesangium, and it is increased in the expanded mesangium in various forms of GN. This paper reports our efforts to investigate the role of Fn in plasma and kidney in patients with GN. Using monoclonal antibodies against human Fn in the ELISA and immunohistoperoxidase techniques to evaluate Fn, we investigated its quantity in connection with clinical state and morphological findings. We studied 93 patients with GN and 26 renal biopsies. The patients with active forms of mesangial proliferative, membranoproliferative and membranous GN showed increased plasma Fn, and the highest levels were in patients with nephrotic syndrome. Increased tissue Fn correlated with mesangial expansion and with IgG and C3 deposits. We speculate on possible mechanisms of the involvement of Fn in human chronic GN.

Fibronectin cell-binding domain triggered transmembrane signal transduction in human monocytes

Fibronectin (Fn) fragments have recently been shown to stimulate tumor necrosis factor (TNF) secretion by human monocytes. In this study, we investigated the signal transduction mechanisms involved in Fn-induced TNF secretion. Treatment of human monocytes with Fn120, a chymotryptic cell-binding fragment of plasma Fn, failed to cause a detectable rise in Ca2+ mobilization. Fn120-induced TNF secretion could be inhibited with Ca2+ channel blockers. The protein kinase C (PKC) inhibitors H-7 and sphingosine inhibited the TNF-inducing activity of Fn120. HA1004 was used as a control for the isoquinoline sulfonamide derivatives and did not change Fn120-induced TNF secretion by monocytes. H-8 inhibited TNF secretion at higher concentrations. A calmodulin-dependent kinase inhibitor, W-7, was found to be effective, with 50% inhibition of Fn120-induced TNF secretion at 5 microM. The activation and translocation of PKC were measured directly. In unstimulated monocytes, approximately 70% of PKC activity was found in the cytosol and 30% in the membrane. Following the stimulation of monocytes with phorbol myristate acetate (100 nM), rapid and sustained translocation of PKC from the cytosol to the membrane was observed. The stimulation of monocytes with Fn120 triggered a rapid translocation of PKC within 2 to 5 min, followed by a return to normal levels within 8 min. These findings support the conclusion that Fn120-induced TNF secretion requires the activation of PKC.

Plasma fibronectin levels during acute rejection and acute tubular necrosis in renal transplant patients.

Fibronectin (Fn), an acute phase glycoprotein synthesized by the liver, has an important immunomodulatory role. We have investigated the changes in plasma Fn in patients after renal transplantation in order to determine whether these changes predict graft injury or rejection episodes. Besides normal healthy controls, healthy pregnant controls, and a trauma control group, we used two groups of chronic renal failure patients as controls: group I, patients with end-stage renal failure (ESRF) on peritoneal dialysis; group II, patients with ESRF on hemodialysis. These were compared with two groups of renal transplant patients: group III, patients 3 months after successful renal transplantation; group IV, patients studied sequentially 10 days immediately posttransplantation. The renal transplant patients were treated with low-dose cyclosporine, azathioprine, and steroids. Citrated plasma samples were collected for Fn assay by a sandwich-type ELISA and for SDS-PAGE analysis and Western blotting. The mean plasma Fn levels were as follows: healthy controls 311.6 SEM, 13.5 micrograms/ml; healthy pregnant controls 357 SEM, 5.9 micrograms/ml; trauma controls 262.3 SEM 31.7, micrograms/ml; group I 169 SEM, 25.1 micrograms/ml; group II 199 SEM, 27.2 micrograms/ml; group III 272 SEM, 21.7 micrograms/ml; group IV 212 SEM, 27.4 micrograms/ml (day 3 postop). There was a significant difference in the plasma Fn levels on day 3 posttransplant between the patients with delayed and immediate renal function (P less than 0.03) (group IV). A significant decrease in plasma Fn levels occurred immediately after steroid therapy was stopped (P less than 0.03) in patients treated for acute rejection. Plasma Fn levels were significantly decreased in the presence of delayed graft function but did not predict rejection.

Fibronectin binding and neutrophil aggregation in burn injury.

Fibronectin (Fn) plays an important role in the adhesive function of many cells including neutrophils (PMN). We examined the hypothesis that activated PMN develop binding sites for fibronectin which allows for the aggregation of contiguous PMN. Because PMN adhesive function is altered in acute burn injury, we also investigated the role of Fn in the aggregation of PMN from subjects with acute thermal injury. The chemotactic peptide, n-formylmethionyl leucyl phenylalanine, induced rapid binding of radioiodinated plasma Fn to PMN. Significant binding of Fn was detected as early as 30 sec poststimulus and maximal binding occurred at 5 min. Fn binding was only partially reversible and nonsaturable. The chemotactic peptide induced aggregation and binding of Fn to PMN with similar kinetics, concentration dependence, temperature, and cation requirements. In burn patients, PMN demonstrated a significant decrease in chemotactic peptide-induced aggregation which was associated with decreased binding of Fn. Alterations in the binding of Fn to PMN may be responsible, in part, for diminished aggregation responses of PMN in the early stages of thermal injury.

Interaction of fibronectin and fibronectin binding protein (FnBP) of Staphylococcus aureus with murine phagocytes and lymphocytes.

In the present study we examined the in vitro and in vivo interactions of a cloned staphylococcal fibronectin binding protein (FnBP) and plasma fibronectin (Fn) with polymorphonuclear cells (PMNs) and macrophages, and how antibodies against FnBP affect the phagocytosis process in vitro. Moreover, the interaction of FnBP and Fn coupled on latex beads as 'artificial bacteria' and 'artificially opsonized bacteria' with murine spleen cells and peritoneal macrophages was tested. The major finding of the present study is that antibodies against the FnBP of Staphylococcus aureus (S. aureus) and low concentration of antibodies recognized two IgG-binding domains of protein A (SpA) are effective in the promotion of phagocytosis in vitro. It was also observed that FnBP has a chemoattractant activity and causes accumulation of PMNs and macrophages in the mouse peritoneal cavity when injected 24 h before irritation of peritoneal exudate. It seems likely that this activity is connected with binding to Fn molecules since formalin inactivation of FnBP (60%) abolished it. In the in vitro phagocytosis assay in the presence of FnBP (in a medium supplemented with serum depleted of Fn), ingestion of bacteria by phagocytes was identical to assay carried out in the presence of BSA. However, addition of plasma fibronectin caused an increased uptake of bacteria by macrophages and to a lesser degree by PMNs. We observed that in a population of normal splenocytes, those cells that effectively bound FnBP- and Fn-coated latex beads were mostly those cells exhibiting macrophage and dendritic morphology. In populations of spleen cells of animal infected with S. aureus, T lymphocytes were also found to bind FnBP- and Fn-coated latex beads. These data suggest that FnBP may have the ability to promote aggregation of immune cells, either directly or by interaction with plasma Fn, which can be helpful in certain cell-cell interactions taking place at the initial stages of specific immune response.

Interaction of fibronectin and fibronectin binding protein (FnBP) of Staphylococcus aureus with murine phagocytes and lymphocytes

In the present study we examined the in vitro and in vivo interactions of a cloned staphylococcal fibronectin binding protein (FnBP) and plasma fibronectin (Fn) with polymorphonuclear cells (PMNs) and macrophages, and how antibodies against FnBP affect the phagocytosis process in vitro. Moreover, the interaction of FnBP and Fn coupled on latex beads as ‘artificial bacteria’ and ‘artificially opsonized bacteria’ with murine spleen cells and peritoneal macrophages was tested. The major finding of the present study is that antibodies against the FnBP of Staphylococcus aureus (S. aureus) and low concentration of antibodies recognized two IgG-binding domains of protein A (SpA) are effective in the promotion of phagocytosis in vitro. It was also observed that FnBP has a chemoattractant activity and causes accumulation of PMNs and macrophages in the mouse peritoneal cavity when injected 24 h before irritation of peritoneal exudate. It seems likely that this activity is connected with binding to Fn molecules since formalin inactivation of FnBP (60%) abolished it. In the in vitro phagocytosis assay in the presence of FnBP (in a medium supplemented with serum depleted of Fn), ingestion of bacteria by phagocytes was identical to assay carried out in the presence of BSA. However, addition of plasma fibronectin caused an increased uptake of bacteria by macrophages and to a lesser degree by PMNs. We observed that in a population of normal splenocytes, those cells that effectively bound FnBP- and Fn-coated latex beads were mostly those cells exbiting macrophage and dendritic morphology. In populations of spleen cells of animals infected with S. aureus, T lymphocytes were also found to bind FnBP- and Fn-coated latex beads. These data suggest that FnBP may have the ability to promote aggregation of immune cells, either directly or by interaction with plasma Fn, which can be helpful in certain cell-cell interactions taking place at the initial stages of specific immune response.

Fibronectin fragments stimulate tumor necrosis factor secretion by human monocytes

Fibronectin (Fn) is a high molecular-weight glycoprotein that can influence many aspects of monocyte function. The purpose of this study was to determine whether Fn could stimulate monocyte tumor necrosis factor (TNF) secretion. Monocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and adherence to plastic (2 h). Plasma Fn was purified from the blood by gelatin-Sepharose affinity chromatography. Monocytes were stimulated with Fn for 18 h and the supernatants were assayed for TNF activity using the L929 bioassay. Intact Fn stimulated the secretion of TNF in a dose-dependent manner. Intact Fn-induced TNF secretion by monocytes was inhibited (50%) but not eliminated by the addition of the R-G-D-containing peptide GRGDSP. Limited proteolysis of the Fn molecule using insoluble chymotrypsin resulted in a fragment preparation that was dramatically more stimulatory than the intact Fn preparation. High-performance liquid chromatographic (HPLC) purification of the fragments demonstrated that at least two fragments were capable of stimulating TNF secretion. Further purification by affinity chromatography and HPLC localized the stimulatory activity to the 120-kd cell-binding fragment. The possibility that the stimulatory activity was the result of endotoxin contamination was ruled out using macrophages from C3H/Hej mice. These results suggest that Fn fragments are potentially important molecules for activation of monocytes and may stimulate monocyte cytotoxic activity.

Glomerulonephritis with organized deposits: A mesangiopathic, not immune complex-mediated disease? A pathologic study of two cases in the same family

Similar glomerular changes (marked widening of the mesangial stalk, irregular basement membrane thickening, and presence of mesangial and subendothelial deposits) were observed by light microscopy in renal biopsy specimens from two patients (mother and daughter) affected by nephrotic syndrome. Electron microscopy disclosed huge glomerular electron-dense deposits containing 12-nm fibrils in both patients. Immunohistochemical investigations performed with antisera anti-immunoglobulin (Ig) and anti-complement fractions, anti-laminin, anti-collagen IV, and anti-fibronectin (FN) showed scant and focal Ig and complement deposits and strong deposits of FN in the mesangium and along glomerular basement membranes. Most glomerular FN was plasma-derived, as shown by immunohistochemical tests with monoclonal antibodies specific for both plasma and cell-derived FN (IST-4) and for cell-derived FN (IST-9). Electron-dense deposits with fibrillar component could hardly correspond to the Ig and complement deposits, whereas they could be related to FN deposits. Since it is known that in glomeruli FN binds to Ig and immune complexes, and the latter seem to be too scant to justify light and electron microscopic lesions and clinical findings, the hypothesis of a primary mesangiopathic glomerulonephritis in some way connected with abnormal plasma FN deposition within the glomeruli and subsequent non-specific immune reactant entrapment could be considered. We could be dealing with a peculiar form of fibrillary glomerulonephritis with rather indolent evolution, as shown by a slow decrease of glomerular function and the scarcely modified glomerular changes found in the second biopsy performed in the mother 8 years after the first investigation.

Fibronectin in plasma and CSF: Evidence for its intrathecal synthesis

A new methodology, using electroimmunodiffusion, has been described for the determination of fibronectin (Fn) in plasma and CSF. Fn was determined in the plasma of 35 normal subjects, and in the plasma and the CSF of 86 patients: 10 controls, 17 definite MS, 15 other inflammatory processes, 11 degenerative diseases, 13 peripheral neuropathies and 20 other neurological diseases. The normal mean was 354 ± 53 μg/ml in plasma, not influenced by sex or age, and in the CSF 2,3 ± 0,85 μg/ml. A significant increase of plasma Fn was observed in each of the patients-group. In the CSF an increase of Fn was observed predominantly in inflammatory processes but not in MS. An intrathecal synthesis of Fn was established in 24 76 patients, most often in inflammatory ( 9 15 ) processes but rarely ( 3 17 ) in MS. The intrathecal synthesis was suspected from the CSF Fn/CSF Albumin ratio and calculated using a formula derived from the principles previously described for IgG intrathecal synthesis. It represents the principal source of Fn increase in the CSF. In all inflammatory processes (including MS) the eventual influence of corticoids, immunosuppresive or anti-inflammatory drugs has not been established. The significance of the CSF Fn level in MS is discussed with regard to the recent demonstration of its presence within MS lesions and on macrophages in plaques.

Human Natural Killer Cells Express Different Integrins and Spread on Fibronectin

Human natural killer (NK) cells adhered and most of them also actively spread on cellular fibronectin (cFn), plasma Fn (pFn) and its Mr 120,000-140,000 or Mr 105,000 cell-binding proteolytic Fn-fragments as well as on heparin-binding Fn-fragments containing the alternative cell binding site. The cells did not spread on vitronectin, laminin or collagens. Adhesion on Mr 105,000 Fn fragment containing the cell binding site, could be prevented by the synthetic peptide GRGDS but not by an inactive peptide, whereas adhesion on heparin-binding Fn fragments was unaffected by the peptide. Spreading of the NK cells led to a distinct reorganization of F-actin. Immunoprecipitation with monoclonal antibodies (MoAb) against the beta 1 integrin subunit of radioactively surface-labelled cells revealed a broad polypeptide band of Mr 140,000 under reducing conditions and a polypeptide doublet of Mr 160,000 and Mr 110,000 under non-reducing conditions. Identical polypeptides, corresponding to the alpha- and beta-subunits of the Fn-receptor complex, were bound to the Mr 105,000 chymotryptic Fn-fragment coupled to Sepharose. Similar experiments with small lymphocytes did not reveal any polypeptides. Immunofluorescence results with McAbs suggested that among the alpha-subunits of integrins, the alpha 3, alpha 4, and alpha 5 subunits are expressed in NK cells. The present results suggest that non-activated NK cells, but not small lymphocytes, express beta 1-integrins, and that at least the Fn-receptors alpha 4 beta 1 and alpha 5 beta 1 may function in the adhesion and migration of NK cells.

Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.

Fibronectin in plasma and CSF: Evidence for its intrathecal synthesis

Abstract A new methodology, using electroimmunodiffusion, has been described for the determination of fibronectin (Fn) in plasma and CSF. Fn was determined in the plasma of 35 normal subjects, and in the plasma and the CSF of 86 patients: 10 controls, 17 definite MS, 15 other inflammatory processes, 11 degenerative diseases, 13 peripheral neuropathies and 20 other neurological diseases. The normal mean was 354 ± 53 μg/ml in plasma, not influenced by sex or age, and in the CSF 2,3 ± 0,85 μg/ml. A significant increase of plasma Fn was observed in each of the patients-group. In the CSF an increase of Fn was observed predominantly in inflammatory processes but not in MS. An intrathecal synthesis of Fn was established in 24 76 patients, most often in inflammatory ( 9 15 ) processes but rarely ( 3 17 ) in MS. The intrathecal synthesis was suspected from the CSF Fn/CSF Albumin ratio and calculated using a formula derived from the principles previously described for IgG intrathecal synthesis. It represents the principal source of Fn increase in the CSF. In all inflammatory processes (including MS) the eventual influence of corticoids, immunosuppresive or anti-inflammatory drugs has not been established. The significance of the CSF Fn level in MS is discussed with regard to the recent demonstration of its presence within MS lesions and on macrophages in plaques.

Fibronectine plasmatique humaine. Comparaison de méthodes de préparation d'un concentré à usage thérapeutique à partir de différentes sources

Three methods, successive precipitations, affinity chromatography on immobilized gelatin and immunoaffinity chromatography with monoclonal anti-fibronectin antibodies were optimized and compared in order to be used for large scale preparation of human plasma fibronectin (Fn). The functional properties of the various Fn preparations were investigated by means of two assays: quantitation of the gelatin-binding activity by ELISA and quantitation of the Fn-mediated attachment of fibroblasts on plastic. Functional alterations of the purified Fn were observed when it was isolated by successive precipitations. Both chromatographic methods provide a rapid and convenient way for isolation of pure and functional Fn. Mass production of monoclonal antibodies is too expensive and legislative requirements for the therapeutic use of monoclonal antibodies are limiting factors for the choice of immunopurification as large scale isolation procedure. Plasma Fn can be isolated from different sources: fresh frozen plasma, cryoprecipitate supernatant or by-products from factor VIII preparation. When gelatin-Sepharose chromatography is performed under optimized conditions, fibronectins isolated from these sources show similar properties. Large scale purifications of Fn from a by-product of factor VIII preparation were performed either by gelatin affinity chromatography or by successive precipitations. These two purification methods can be easily scaled-up since the data obtained closely correlate with analytical results. The chromatographic method supplies a higher purified (98 vs 75%) and functional (95 vs 50%) material when compared with successive precipitations. Yield is also higher (50 vs 26%). The starting material undergoes viral inactivation and the affinity purified Fn, sterile, atoxic, apyrogen, which can be freeze-dried without additives fulfils all requirements for an injectable product.