Eshin Cho

Reversal of fibronectin and opsonic deficiency in patients. A controlled study.

Plasma fibronectin is an opsonic glycoprotein which augments reticuloendothelial phagocytic clearance of nonbacterial participates. We evaluated the influence of intravenous infusion of plasma cryoprecipitate on circulating immunoreactive fibronectin and associated opsonic activity at 0.5, 2.0, 4.0, 10, and 21 hr postinfusion in septic (n = 8) and nonseptic (n = 6) surgical and/or trauma patients with documented plasma fibronectin deficiency. The study was a randomized, double-blind, crossover clinical protocol in which fibronectin-poor (0.116 ± 0.025 mg/ ml) cryoprecipitate extracted plasma (placebo) was compared to fibronectin-rich (2.139 ± 0.161 mg/ml) plasma cryoprecipitate. Septic injured patients (149.37 ± 17.11 μg/ml) had lower (p < 0.05) plasma fibronectin levels than nonseptic injured patients (212.17 ± 7.14 μg/ml) and both were less (p < 0.05) than normal (330 ± 30 μg/ml). As tested in vitro with a peritoneal macrophage monolayer assay, cryoprecipitate manifested opsonic activity related to its fibronectin concentration. Intravenous infusion of fibronectin rich cryoprecipitate reversed both the immunoreactive fibronectin and opsonic deficiency, while infusion of the placebo at a comparable total protein load did not reverse either deficient parameter. Reversal of fibronectin deficiency was more sustained in nonseptic injured patients as compared to septic injured patients. Thus, reversal of opsonic deficiency in septic and nonseptic injured patients is observed after infusion of plasma cryoprecipitate and not with infusion of fibronectin deficient plasma at comparable protein loads. Also, cryoprecipitate extracted plasma may serve as an appropriate control solution for randomized studies evaluating the therapeutic value of fibronectin-rich plasma cryoprecipitate.

Opsonic fibronectin deficiency in the etiology of starvation-induced reticuloendothelial phagocytic dysfunction

The role of nutritional deficiencies in mediating host defense failure is increasingly recognized. This study evaluated the effect of starvation on serum opsonic fibronectin levels and reticuloendothelial function in the rat. Over a period of 5 days of starvation serum immunoreactive opsonic fibronectin had fallen to 62% of control levels (458 ± 20 μg/ml). This correlated closely (r = 0.85, P < 0.05) with a fall in bioassayable opsonic activity. Addition of purified opsonic fibronectin to plasma obtained from starved rats restored its ability to stimulate in vitro Kupffer cell phagocytic activity. Reticuloendothelial phagocytic function in vivo was significantly depressed after 5 days starvation as reflected by prolonged blood retention and decreased hepatic Kupffer cell uptake of a test colloid. Intravenous fibronectin therapy (2 mg/100 g), 1 hr prior to assessment of RE function, significantly (P < 0.05) improved but did not fully normalize blood clearance and hepatic uptake. These data suggest the RES phagocytic host defense failure in the nutritionally compromised patient may occur, in part, as a consequence of opsonic fibronectin deficiency.

Decreased Plasma Fibronectin during Starvation in Man

This study evaluated the effect of 5 days of starvation followed by 5 days of refeeding on immunoreactive plasma and serum fibronectin and associated opsonic activity as studied by peritoneal macrophage monolayer bioassay in 12 healthy women volunteers. The temporal alteration of fibronectin was compared with the serum albumin, total iron-binding capacity, and retinol-binding protein levels. Fibronectin concentration and opsonic activity were also determined in two cachectic patients who were 61 and 78% of their ideal body weight. Prior to starvation, plasma fibronectin was 292 +/- 20 micrograms/ml and serum fibronectin was 182 +/- 16 in all subjects. After 5 days of starvation, immunoreactive fibronectin decreased (p less than 0.05) by 20-25%. This decrease was not great enough to impair opsonic activity as tested by the in vitro macrophage assay. Starvation caused no decrease in serum albumin or total iron-binding capacity, although retinol-binding protein decreased by 35%. During refeeding, subjects were randomized to a diet with (n = 6) and without (n = 6) carbohydrate. After 5 days of refeeding, fibronectin levels were normalized on the carbohydrate-containing diet, but were still low (82% of normal) on the carbohydrate-free diet. Retinol-binding protein did not fully normalize after 5 days of refeeding. In the two cachectic patients, fibronectin levels prior to total parenteral nutrition were 25 and 75% of normal. Thus, starvation can lower fibronectin levels and this protein is rapidly restored with adequate nutrition.

Reversal of opsonic deficiency in surgical, trauma, and burn patients by infusion of purified human plasma fibronectin. Correlation with experimental observations

Plasma fibronectin deficiency has been documented in critically ill surgical, trauma, and burn patients. Human plasma fibronectin was isolated by gelatin-Sepharose affinity chromatography and evaluated with respect to its opsonic activity following pasteurization, its in vivo clearance kinetics, and its short-term influence on cardiovascular hemodynamics in postoperative septic sheep. Six patients with low plasma fibronectin levels were also evaluated with respect to temporal changes of immunoreactive fibronectin and opsonic activity following infusion of fibronectin at a dose calculated to elevate the plasma fibronectin level to 400 micrograms/ml. With utilization of three different in vitro radioisotopic phagocytic assays, i.e., liver slice assay, peritoneal macrophage monolayer assay, and Kupffer cell monolayer assay, retention of opsonic activity by fibronectin following pasteurization was documented. The normal biphasic kinetics associated with plasma clearance of fibronectin were also not altered by pasteurization. In postoperative septic sheep with hemodynamic instability, intravenous infusion of 500 mg of purified human fibronectin initiated no abnormal hemodynamic response. Indeed, as compared with placebo, the infusion of fibronectin into the postoperative septic sheep resulted in a more stable systemic vascular resistance and pulmonary vascular resistance with a higher arterial pressure. It also elevated immunoreactive fibronectin levels (p less than 0.05) and increased opsonic activity (p less than 0.05). Surgical, trauma, and burn patients (ages 18 to 80 years) with low plasma fibronectin levels (160 to 236 micrograms/ml) manifested no disturbance in cardiovascular, respiratory, or hematologic parameters following fibronectin infusion (590 to 988 mg per patient), but did display an early increase of opsonic activity. This standardized, pasteurized, and opsonically active preparation of purified human plasma fibronectin (5.0 mg/ml after reconstitution) has utility for future randomized clinical trials in injured patients with sepsis.

Normal Fibronectin Levels as a Function of Age in the Pediatric Population

Summary: Fibronectin is an important non-immune opsonic protein influencing phagocytic clearance of blood-borne nonbacterial particulates which may arise in association with septic shock, tissue injury, and intravascular coagulation. In the present study, serum fibronectin was measured by both electroimmunoassay as well as rapid immunoturbidimetric assay in healthy children (n = 114) ranging in age from 1 month to 15 years in order to delineate the temporal alterations in fibronectin with age. Normal adult serum fibronectin concentrations are typically 220 μg/ml ± 20 μg/ml. Serum concentration is 35–40% lower than normal plasma concentration due to the binding of fibronectin to fibrin during clot formation. Children between 1–12 months of age bad significantly (P < 0.05) lower serum fibronectin levels than children between the ages of 1–15 years. Progressive elevation in fibronectin levels was observed within the last 8 months of the first year of age. Fibronectin levels in children older than 1 year of age remained constant up to 15 years and were within the lower limit of the normal adult concentration. No significant (P > 0.05) difference in serum fibronectin was observed between male and female children at all age groups. Fibronectin levels thus, increase during the first year of age and normal levels of this blood protein in the infant are less than the normal range for adults.

Liver and spleen phagocytic depression after peripheral ischemia and reperfusion

Liver and spleen phagocytic clearance of blood-borne microparticulate tissue debris and products of intravascular coagulation after trauma and surgical injury is an important mechanism to limit the deposition of debris in the pulmonary vascular bed. Plasma fibronectin (pFn) modulates this clearance process. We evaluated the effect of a localized peripheral ischemia and reperfusion injury on liver and spleen phagocytic function. Male rats (250 to 350 g) underwent 4 hours of tourniquet-induced bilateral hindlimb ischemia, followed by 18 hours of reperfusion after release of the tourniquet. Rats subjected to ether anesthesia alone or anesthesia followed by groin incision without ischemia were the control and sham groups, respectively. Reticuloendothelial (RE) phagocytic function was assessed at 15 minutes and 18 hours after the start of reperfusion by the in vivo liver and spleen removal of blood-borne iodine 125 (125I)-test microparticles, which were coated with gelatin (denatured collagen) to enhance their interaction with pFn. Liver and spleen particle uptake in control and sham rats was similar. In contrast, after 4 hours of ischemic injury with 15 minutes of reperfusion, we observed a 30% to 40% decrease (p less than 0.05) in liver and spleen particle uptake as compared with sham controls with partial restoration of this removal mechanism by 18 hours. This depression in liver and spleen phagocytic function was associated with a significant (p less than 0.05) increase in the deposition of the 125I-test particles in the lung. RE depression was not due to a deficiency of pFn; indeed, a marked elevation (588 +/- 12 micrograms/mL versus 1,083 +/- 40 micrograms/mL) of pFn was observed by immunoassay over the 18-hour reperfusion interval. Comparative bioassay of humoral (opsonic) versus cellular (Kupffers cell) activity revealed that Kupffers cells in livers from controls or ischemia-reperfusion rats exhibited normal phagocytic function when incubated in plasma harvested from either control or 4-hour ischemic rats. The opsonic activity of plasma harvested after ischemia and reperfusion was also more than adequate, consistent with the immunoassay analysis. Thus, the impaired liver and spleen clearance mechanism after peripheral ischemia and reperfusion injury did not appear to be due to either a macrophage cellular deficit or a lack of pFn. This clearance depression may be mediated by splanchnic malperfusion, which is known to develop after peripheral ischemia and reperfusion and associated soft tissue injury.

Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.

Fragmentation of subendothelial matrix‐bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125l‐labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I‐fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose‐dependent manner by the addition of both methoxysuccinyl‐alanine‐alanine‐valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non‐specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60–200 kD) were not labelled, but the smaller molecular weight fragments (<45 kD), derived from the artifical matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.

Hepatic removal of 125I-DLT gelatin after burn injury: a model of soluble collagenous debris that interacts with plasma fibronectin.

The decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin‐coated particles to simulate blood‐borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 1251‐labeled dilactitol tyramine (DLT‐gelatin) as a model of soluble collagenous tissue debris. We studied its blood clearance as well as organ localization in normal and postburn rats. Fibronectin‐deficient plasma harvested early after burn exhibited limited ability to support in vitro phagocytic uptake of the gelatinized microparticles by Kupffer cells in liver tissue from normal rats. However, Kupffer cells in liver tissue from normal and postburn rats phagocytized the test particles at a normal rate when incubated in normal plasma. The DLT‐gelatin ligand bound to fibronectin in a dose‐ dependent manner as verified by its capture with anti‐ fibronectin coated plastic wells when coincubated with purified fibronectin. By gel filtration chromatography, the binding of fibronectin with the DLT‐gelatin ligand was readily detected, resulting in the formation of a high‐ molecular‐weight complex. In normal animals the plasma clearance and liver localization of 125I‐DLT‐gelatin was competitively inhibited by infusion of excess nonradioactive gelatin. The blood clearance and liver localization of the soluble gelatin ligand were also impaired after burn injury during periods of fibronectin deficiency similarly to the pattern observed with gelatin‐coated microparticles. By autoradiography, the cellular site for the uptake of the 1251‐DLT‐gelatin was primarily but not exclusively hepatic Kupffer cells; 125I‐DLT‐asialofetuin and 125I‐ DLT‐ovalbumin were removed by hepatocytes and sinusoidal endothelial cells, respectively. Thus, gelatin conjugated with 125I‐DLT can be used to simulate blood‐ borne soluble collagenous tissue debris after burn. It rapidly binds to plasma fibronectin before its hepatic Kupffer cell removal, and its blood clearance is markedly delayed after burn injury during periods of plasma fibronectin deficiency.

Effect of acute plasma fibronectin depletion on tissue fibronectin levels: Analysis by a new fluorescent immunoassay

Plasma fibronectin modulates reticuloendothelial (RE) phagocytosis of cellular and tissue debris, fibrin microaggregates, and gelatin-coated particulates. An antigenically related, but more insoluble form of fibronectin is found in various tissues and suspected to play a role in vascular permeability, cell adhesion, and wound healing. The current study developed a fluorescent immunoassay which could be utilized for the quantification of tissue fibronectin following its extraction from tissues. Additionally, the changes in tissue fibronectin induced by the intravenous injection of gelatin-coated colloids in rats (300-350 g), which acutely depletes the plasma fibronectin level, were also investigated. Injection of gelatinized RE test lipid emulsion (50 mg/100 g) depleted the plasma fibronectin at 2 hr (80-85% depletion) followed by rebound restoration within 24 hr as quantified by either electroimmunoassay or fluorescent immunoassay. RES system clearance of the test particles from the blood resulted in an acute elevation in fibronectin extractable from the liver with a normalization by 48 hr. In contrast, assay of tissue fibronectin following a single extraction revealed a decrease in lung extractable fibronectin within 2 hr following RE blockade which persisted for 24-48 hr. Extractable fibronectin in spleen and renal tissue was unaltered by RE blockade. This microfluorescent immunoassay may provide a sensitive method to quantify fibronectin in small aliquots of tissue. Increased hepatic fibronectin most likely reflects interiorization of plasma fibronectin during Kupffer cell clearance of the test particles. Decreased lung extractable fibronectin may alter lung vascular sensitivity to a subsequent septic and/or intravascular coagulation stress. Thus, similar to the labile nature of plasma fibronectin, the concentration of fibronectin in various tissues, can undergo dynamic alterations.

Factor XIII as a modulator of plasma fibronectin alterations during experimental bacteremia.

Fibronectin is found in plasma as well as in association with connective tissue and cell surfaces. Depletion of plasma fibronectin is often observed in septic trauma and burned patients, while experimental rats often manifest hyperfibronectinemia with sepsis. Since Factor XIII may influence the rate of clearance and deposition of plasma fibronectin into tissues, we evaluated the temporal changes in plasma fibronectin and plasma Factor XIII following bacteremia and RE blockade in rats in an attempt to understand the mechanism leading to elevation of fibronectin levels in bacteremic rats, which is distinct from that observed with RE blockade. Clearance of exogenously administered fibronectin after bacteremia was also determined. Rats received either saline, Pseudomonas aeruginosa (1 X 10(9) organisms), gelatinized RE test lipid emulsion (50 mg/100 gm B.W.), or emulsion followed by Pseudomonas. Plasma fibronectin and Factor XIII were determined at 0, 2, 24, and 48 hours post-blockade or bacteremia. At 24 and 48 hr following bacteremia alone or bacteremia after RE blockade, there was a significant elevation (p less than 0.05) of plasma fibronectin and a concomitant decrease (p less than 0.05) of plasma factor XIII activity. Extractable tissue fibronectin from liver and spleen was also increased at 24 and 48 hours following R.E. blockade plus bacteremia. In addition, the plasma clearance of human fibronectin was significantly prolonged (p less than 0.05) following bacterial challenge. Infusion of activated Factor XIII (20 units/rat) during a period of hyperfibronectinemia (908.0 +/- 55.1 micrograms/ml) resulted in a significant (p less than 0.05) decrease in plasma fibronectin (548.5 +/- 49.9 micrograms/ml) within 30 min. Thus Factor XIII deficiency in rats with bacteremia may contribute to the elevation in plasma fibronectin by altering kinetics associated with the clearance of fibronectin from the blood.