Immunofluorescent Analysis of Plasma Fibronectin Incorporation into the Lung during Acute Inflammatory Vascular Injury

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Abstract
Incorporation of plasma fibronectin into tissues is believed to influence endothelial cell-cell interaction, as well as endothelial cell adhesion to matrix. We used immunofluorescent microscopy coupled with tissue extraction of noncovalently incorporated fibronectin to delineate the time course for matrix incorporation of soluble plasma-derived fibronectin into the lung of sheep during postoperative bacteremia. Adult sheep were surgically prepared with both lung and peripheral lymph fistulas. Sheep were anesthetized 2 days following surgery and injected intravenously with a sublethal dose of live Pseudomonas aeruginosa, which consisted of 5 × 108 live organisms suspended in 0.9% saline. Bacterial infusion elicited a 300% increase in lung transvascular protein clearance but no increase in peripheral transvascular protein clearance. Purified dimeric human plasma fibronectin (hFn), used as an “immunologic marker,” was then infused intravenously (100 mg/sheep) into two additional groups of sheep (nonbacteremic control group and bacteremic experimental group) and allowed to mix with the plasma pool of endogenous soluble sheep fibronectin (sFn). Incorporation of the plasma-derived hFn into the lung matrix and its distribution in relation to endogenous sheep fibronectin in the matrix was assessed by dual-label immunofluorescence using antibodies specific to either sFn or hFn. Human fibronectin from the vascular compartment codistributed with endogenous sheep fibronectin in the lung matrix. Moreover, its deposition into the lung was markedly increased in postoperative bacteremic sheep compared with nonbacteremic control sheep. Increased hFn deposition in the lung with bacteremia was clearly apparent within 2 h. The hFn deposited in the lung was nonextractable using a heparin-urea tissue extraction buffer, suggesting its rapid covalent cross-linking and incorporation into the lung matrix. Microscopic analysis of serial lung biopsies revealed focal areas of inflammation with an intense mononuclear infiltrate into the lungs by 2 h in the bacteremic sheep. Interstitial edema and vascular endothelial injury were observed by 4 h, with alveolar edema apparent over 6 to 8 h. Thus, postoperative bacteremia results in a rapid incorporation of plasma fibronectin into the lung matrix. This may be a physiologic mechanism to stabilize the integrity of the lung vascular barrier.