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Fibronectin is a normal glycoprotein component of plasma, interstitial fluid, and extracellular matrix which has binding sites for collagen, gelatin, actin, glycosaminoglycans, fibrin, Staphylococcus aureus, and some cells. Since it is a dimer, it can crosslink these substances to each other or to extracellular components of basement membrane, thereby affecting many physiological processes. The level of circulating fibronectin is markedly reduced following even moderate blunt or operative trauma, thermal injury, starvation, advanced cancers, hemorrhage, etc. Replacement therapy has been tried with some success in patients who become septic following multiple injuries. The reduction in plasma fibronectin has been attributed to several causes including consumption by binding to cell debris at the site of injury, binding to circulating cell debris and its subsequent removal by elements of the phagocytic system, and degradation by proteolytic cleavage. However, the amount of fibronectin removed from circulation raises some question about this. In this paper, we used indomethacin, ibuprofen, imidazole, and essential fatty acid deprivation to inhibit the synthesis of prostaglandins in young adult rats. Thirty minutes after ip administration of one of the inhibitors, the rats were subjected to a midline laparotomy and mild intestinal manipulation. Blood samples were taken at intervals following closure of the incision and analyzed for fibronectin. In all cases, the normal decline in plasma fibronectin seen in untreated rats was abrogated by inhibiting prostaglandin synthesis. Since imidazole specifically inhibits thromboxane A synthesis, this strongly suggests that thromboxanes directly or indirectly control the trauma-induced reduction in circulating fibronectin. This was confirmed by ip injection of thromboxane into the rats which resulted in a decline in plasma fibronectin levels.

Fibronectin (Fn), a normal serum protein which appears to have important roles in wound healing and in reticuloendothelial system function, is depressed by most types of trauma. Fn is released into the tissue at the site of an injury which suggests the depression is the result of Fn sequestration at the wound site. A competitive inhibition assay for Fn was used to measure the concentration of Fn in fluid draining the site of a radical mastectomy and the level in concurrently obtained plasma. Plasma levels of Fn were significantly depressed following surgery but were returning toward normal by 24 hours postsurgery. The concentration of Fn in drainage fluid collected two hours postop was slightly but significantly lower than the plasma collected simultaneously. By 8 hours after surgery, drainage fluid levels were significantly higher than that in concurrently obtained plasma, and the difference was even more pronounced at 24 hours postop. Fn in the drainage fluid retained opsonic activity but at a lower level than the opsonic activity in plasma. The higher concentration of Fn in drainage fluid than in plasma appears to be due to binding of the Fn to tissue debris in the exudate, which prevents the reentry of Fn into the vascular compartment.

Cold insoluble globulin (fibronectin, alpha 2-surface binding glycoprotein) is a naturally occurring substance necessary for optimal stimulation of the reticuloendothelial system. While this globulin depends on macrophages as the effector cells for its opsonic function, as is true of both antibody and complement, it is neither part of nor dependent on these systems for its opsonic activity. A relatively simple bioassay developed at the Medical College of Georgia substantiated that cold insoluble globulin is severely depleted in sepsis. Cryoprecipitate, properly processed and stored, is an exogenous source of cold insoluble globulin. Infused into septic patients 10 units thawed at 2 degrees C and reconstituted to 250 ml with saline solution can temporarily restore cold insoluble globulin levels and enhance activity of the reticuloendothelial system. Proper current use dictates measurement of cold insoluble globulin levels in the infusate as well as levels in the patient and the clinical response to infusion. Our bioassay and a septic patient's response to infusion of cold insoluble globulin are reported herein.

Fibronectin (Fn) is a normal plasma and extracellular matrix glycoprotein that is thought to be important in reticuloendothelial system function as well as in promoting adhesion of various cell types to basement membranes and to each other. Plasma Fn levels are depressed following almost any type of trauma. It opsonizes circulating tissue debris for removal by the fixed cells of the reticuloendothelial system. It has been assumed but not proven that Fn also opsonizes tissue debris at the site of the injury for subsequent phagocytosis by tissue macrophages. In this study, rats were given intracardiac injections of Fn coupled with fluorescence isothiocyanate (Fn-FITC) and human serum albumin-rhodamine isothiocyanate (HSA-RITC). Abdominal Rebuck skin windows were then prepared. Within 24 hours, debris at the sites of injury were observed to be coated with Fn-FITC but not HSA-RITC. This Fn-labeled debris was subsequently ingested by macrophages at the site. No macrophages were found that had taken up HSA-RITC. Thus, Fn is seen to coat tissue debris and effete cells within the wound, and the coated material is subsequently removed by tissue macrophages.

Fibronectin (Fn) synergizes with macrophages (M phi) in inducing cytostasis and cytotoxicity of neoplastic cells in culture. Since heparin enhances Fn's opsonic activity in many systems, we investigated its effect on Fn-macrophage synergy in cytostasis. MCG-T14 (a spontaneous mouse mammary adenocarcinoma) cells (4 X 10(4] were added to wells both with and without C. parvum activated M phi monolayers. To these cultures were added increasing concentrations of Fn with or without heparin. Fn synergizes with both M phi S and heparin in inhibiting tumor cell growth. The combined cytostatic effect of Fn, heparin and M phi S is more than additive. In other experiments, MCG-T14 cells were pre-incubated for 2 hr with Fn, washed free of Fn, and treated as above. The results of these experiments were similar to coculture experiments, but the effect of heparin was even more pronounced. Dermatan sulfate and hyaluronic acid had a variable effect on Fn and Fn-macrophage induced cytostasis.

Purified fibronectin (Fn) mediated attachment of MCG-T14 cells, a mouse adenocarcinoma, to culture vessel surfaces. Concentrations of Fn less than 30 micrograms/ml enhanced the growth rate of these cells as judged by 3H-thymidine incorporation, whereas higher levels of Fn were inhibitory. Concentrations of Fn and macrophages, which had little or no effect on the growth rate of the T14 cells when added individually, mediated a 64% decrease in the rate of growth of these cells when cultured together. Free Fn was not required for this effect since target cells pretreated with Fn and then washed also were susceptible to growth inhibition by macrophages. These results indicate that Fn is able to mediate an enhancement of macrophage antitumor activity, probably by supporting the binding of target cells to the macrophage effector cells.

Fibronectin (Fn) is a normal plasma and extracellular matrix glycoprotein that is involved in each phase of wound healing. For example, it is incorporated into both fibrin and collagen fibers; it opsonizes circulating tissue debris for removal by the reticuloendothelial system; it is used by macrophages, fibroblasts, and epithelial cells to move into the wound; and fragments of Fn are chemotactic for fibroblasts. In this study, experiments with rats showed that excised lesions treated with Fn healed more rapidly than paired control lesions treated with the carrier alone. Applications of Fn once a day for two days were as effective in speeding healing as twice-daily applications of Fn for 12 days. A single treatment with Fn soon after the initial injury was nearly as effective as more prolonged treatment regimens.

The effects of cold insoluble globulin (CIG) on short-term cultures of Walker 256 carcinoma cells have been determined. Carcinoma cell proliferation in the presence of normal or heat-treated (opsonically inactive) rat serum, affinity-purified CIG, albumin, or medium alone was measured by determination of [3H]TdR incorporation into cellular DNA and by actual cell counts. The addition of serum or affinity-purified CIG significantly reduced proliferation compared with that which occurred in the presence of albumin or medium alone. The reduced proliferation caused by CIG is due to an inhibition of cell growth (cytostasis) rather than to cell death (cytotoxicity) as demonstrated by a lack of [125I]UdR release from prelabeled carcinoma cells incubated with CIG.

Macrophages are important positive and negative regulators of both primary and secondary antibody responses, and their activity may, in turn, be controlled by soluble mediators secreted by other cells. Fibronectin is a 440,000 dalton normal constituent of plasma and extracellular membranes that acts through macrophages to inhibit mitogen- and alloantigen-stimulated lymphoproliferation. We examined the effect of Fn on the antigen-stimulated lymphoproliferative and antibody responses in cells from trinitrophenol-derivitized keyhole limpet hemocyanin (TNP-KHL) primed rats. Fn in concentrations equivalent to normal plasma levels inhibited TNP-KLH-stimulated lymphoproliferation by unseparated lymph node leukocytes. When the experiment was repeated using purified lymph node T cells and added thioglycollate-induced peritoneal exudate macrophages or splenic adherent macrophages, Fn alone and TNP-KLH alone stimulated lymphoproliferation, but in combination they were strongly inhibitory. The effect was not due to decreased lymphocyte viability in the presence of both TNP-KLH and Fn. Nor was it due to complexes between TNP-KLH and Fn or to a simple alteration in the kinetics of lymphoproliferation. Fn had to be present with the TNP-KLH within the 1st hour of incubation. If macrophages were coincubated with TNP-KLH and Fn for 24 h, washed, and added to enriched T cells, inhibition was equivalent to that seen with continuous coculture. Similarly, coculture of TNP-KLH and Fn inhibited both total immunoglobulin and TNP-KLH-specific antibody synthesis at optimal concentrations of splenic adherent cells. However, at suboptimal levels of splenic macrophages, the combination was synergistic, stimulating more total immunoglobulin synthesis than either TNP-KLH or Fn alone. These data suggest that the inhibitory effect was dependent upon the concentration and phenotype of macrophages present in culture.

Levels of cold insoluble globulin (CIG), also called opsonic alpha2 surface-binding glycoprotein, humoral recognition factor, and fibronectin, are depressed as a result of major trauma. This protein normally promotes the phagocytosis and removal of abnormal particles and bacteria by cells of the recticuloendothelial system. Although CIG depends on macrophages as the effector cells for its opsonic function as is true of both antibody and complement, CIG is neither part of nor dependent on these systems for its opsonic activity. Using a new, rapid bioassay, we have demonstrated that in humans subjected to operative trauma, circulating CIG levels are considerably depressed in the early hours after operation and return to normal within 24 hours. The observed decreases in titer varied in extent, time of onset, and duration. Continuing study is designed to determine potential correlation between initial levels of CIG, decreases in titer resulting from operation, and the incidence of postoperative infection.

A competitive inhibition assay for functional fibronectin (Fn), based on ELISA technology, is described. The assay measures Fn's physiologic ability to bind to denatured collagen (gelatin). Affinity-purified Fn inhibits the binding of alkaline phosphatase coupled Fn to gelatin-coated wells of a microtiter plate in a concentration-dependent manner. The assay range is 50-500 micrograms Fn/ml, which is suitable for the measurement of plasma Fn in both normal and opsonin deficient individuals. It is reproducible over an eightfold dilution of plasma and is resistant to interference by normal plasma proteins. The assay described is quick, quantitative, and reproducible, and satisfies the need for a measure of functional Fn activity in the clinical laboratory.