Silica induced suppression of the production of third and fifth components of the complement system by human lung cells in vitro.

Although investigations to date have demonstrated the ability of the monocyte/macrophage to synthesize complement components, only a limited number of studies on complement synthesis by nonhepatic tissue cells have been reported. To begin to fill this gap in our knowledge we have recently evaluated the ability of lung tissue cells to synthesize and secrete various complement components in vitro. Using 35S-methionine incorporation and immunoprecipitation techniques we have previously demonstrated the ability human lung type II pneumocytes (A549) and human lung fibroblasts (WI-38), to synthesize and secrete a variety of both early and terminal complement components, as well as several regulatory proteins including Clr, Cls, C4, C3, C5, C6, C7, C8, C9, Factor B, Factor H, Factor I and Cls inactivator. Our present studies demonstrate the capability of silica to regulate complement component production by A549 cells, but not complement component production by WI-38 cells. Specifically, using sensitive ELISAs we demonstrated that a non-toxic dose of silica had the capability to suppress the production of both C3 and C5 by A549 pneumocytes by 40-50 percent, but had no effect on C3 or C5 synthesis by WI-38 fibroblasts. Additionally, using 35S-methionine incorporation and TCA precipitation techniques, we demonstrated that suppression of C3 and C5 production by silica treated A549 pneumocytes was not a result of suppression of total protein synthesis. These studies demonstrate that silica, which has been implicated in pulmonary diseases, has the capability to regulate local complement production by lung tissue cells in vitro. In vivo, this suppression of complement production by the type II pneumocytes could alter the local tissue reservoir of complement components during infection and pulmonary injury, thus resulting in depressed pulmonary host defense.

Cytokine regulation of C3 and C5 production by human corneal fibroblasts.

Recent investigations have suggested that cytokines play important roles during inflammation and host defense, primarily by regulating the diverse functions of immunologic cells (e.g. lymphocytes and monocytes). However, much less is known about the capacity of cytokines to also regulate the functions of resident tissue cells. We hypothesize that during inflammation, cytokines (e.g. monokines and lymphokines) directly regulate the expression of inflammatory precursors and mediators, such as the third and fifth complement components, by resident ocular cells and are therefore important in the local regulation of ocular inflammation. To test this hypothesis we developed an in vitro culture system utilizing isolated human corneal fibroblasts and examined the effects of specific cytokines, i.e. interleukins and interferons, on the production of the third and fifth components of the complement system. Human corneal fibroblasts were cultured in the presence of varying concentrations (1-500 U ml-1) of interleukin 1 alpha, interleukin 1 beta, interleukin 2, interferon alpha and interferon gamma for 48 hr at 37 degrees C, 5% CO2. The supernatants were then evaluated for antigen levels for the third and fifth components of complement using specific enzyme-linked immunospecific assays. These studies revealed that both interleukin 1 alpha and interleukin 1 beta induced seven to tenfold increases in the levels of the third component. Similarly interferon alpha and interferon gamma stimulated an approximate four and ninefold dose-dependent increase, respectively, in the production of the third component. Analysis of the effect of interleukin 2 on third component production demonstrated that higher concentrations (100 U ml-1) were required to induce a fivefold increase in the production of the third component.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vitro model of cell migration: evaluation of vascular endothelial cell migration.

In vivo vascular endothelial cell (VEC) migration is thought to play a central role in the development of new capillaries as well as the resurfacing of large vessels. Recently, we have developed an in vitro VEC migration assay system based on the ability of VEC to migrate off of tissue culture microcarrier beads. For these studies, bovine pulmonary artery VEC were grown to confluence on Cytodex 3 microcarrier beads (MCB). Next, the confluent VEC covered microcarrier beads were pipetted into 4-cm2 wells of a tissue culture plate and incubated at 37 degrees C/5% CO2. At various time intervals, the movement of the VEC off of the MCB onto the tissue culture surface was evaluated microscopically. Using this assay, we have studied the effect of endothelial cell growth supplement and various matrices (i.e., fibronectin, gelatin, and Matrigel) on VEC migration. These studies demonstrated that: (i) gelatin had no effect on normal or mitomycin C-pretreated VEC migration; (ii) fibronectin had no effect on normal VEC migration, but stimulated the relative migration of mitomycin pretreated VEC; and (iii) Matrigel significantly suppressed both normal and mitomycin C-pretreated VEC migration. Endothelial cell growth supplement (ECGS) stimulated both normal and mitomycin C-pretreated VEC migration on fibronectin at concentrations of 10 micrograms/ml ECGS. Pretreatment with ECGS had no effect of normal or mitomycin C VEC migration on gelatin. Finally, ECGS stimulated a statistically significant increase in the migration of normal and mitomycin C-pretreated VEC migration on Matrigel.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine regulation of C3 and C5 production by the human type II pneumocyte cell line, A549.

A growing body of literature suggests that a variety of cell products (e.g., cytokines, C components, etc.) likely play an important role during inflammation and host defense by locally regulating the diverse functions of recruited (i.e., immunologic cells) as well as tissue cells. Previously, a number of investigations have demonstrated the ability of immunologic cells to produce C components in vitro, and further studies have identified a variety of cytokines that can regulate C component production by these cells. Recently, we have demonstrated the ability of lung tissue cells, including epithelial cells and fibroblasts, to synthesize and secrete numerous C components and complement regulatory proteins in vitro. Additionally, we have demonstrated that C component production can be modulated by a variety of factors including endotoxin and serum. In our studies we investigated the effects of specific cytokines, i.e., IL and IFN, on the production of the third (C3) and fifth (C5) C components by the continuous cell line of human type II pneumocytes (A549). Specifically, using sensitive ELISA we demonstrated that A549 pneumocytes exposed to IL-1 alpha, IL-1 beta, or IL-2 induced a dose-dependent, more than twofold, increase in C3 production and a 50% decrease in C5 production when compared to control (untreated) A549 cells. Interestingly, IFN-alpha significantly decreased both C3 and C5 production, i.e., 38 and 71%, respectively, in a dose-dependent manner. IFN-gamma had no effect on C3 production, but significantly decreased C5 production by A549 pneumocytes by 84%. These data not only demonstrate that cytokines have the capability to modulate C3 and C5 production by human type II pneumocytes in vitro, but that C3 and C5 production by these cells can be regulated independently by different cytokines. In vivo, cytokine modulation of C component production by local tissue cells likely plays an important role in the regulation of inflammation and host defense within the lung.

Effect of lipopolysaccharide on C3 and C5 production by human lung cells.

Although studies to date have demonstrated the ability of the monocyte/macrophage to produce C components in vitro, very few studies on C production by nonhepatic tissue cells have been reported. Recently, using 35S-methionine incorporation and immunoprecipitation techniques our laboratory has demonstrated the ability of tissue cells, i.e., the human lung type II pneumocyte (A549) and human lung fibroblast (WI-38), to synthesize and secrete a variety of early and terminal complement components, as well as several regulatory proteins in vitro, i.e., C1r, C1s, C4, C3, C5, C6, C7, C8, C9, factor B, factor H, factor I, and C1s inactivator. In our studies, we extended these observations by demonstrating the capability of LPS to modulate C3 production by A549 pneumocytes. Specifically, using a sensitive ELISA we demonstrated that A549 pneumocytes exposed to LPS induced an 80 to 180% increase in C3 levels when compared to untreated A549 cells. Interestingly, LPS had no effect on C5 production or total protein synthesis by A549 pneumocytes. In the case of the WI-38 fibroblast, LPS had no effect on 1) C3 production, 2) C5 production, or 3) total protein synthesis in vitro. These studies demonstrate that agents such as LPS have the potential to selectively regulate C production (i.e., C3) in individual lung cells in vitro, and suggests that in vivo LPS may alter the local tissue reservoir of C components during infection and lung injury, thus impacting on pulmonary inflammation and host defense.

Characterization of the anaphylatoxin inactivator and chemotactic factor inactivator activities during cardiopulmonary bypass.

Complement activation productive of phlogistic products has been suggested as one of the major mechanisms of the pump lung syndrome associated with cardiopulmonary bypass (CPB) surgery. Recent studies have demonstrated the presence of circulating C3a antigens in the serum of patients undergoing CPB and have suggested that the vasoactive nature of C3a may contribute directly to the interstitial edema and vascular changes seen in pump lung syndrome. In an effort to unravel the underlying mechanisms of pump lung syndrome, we undertook investigations to determine whether CPB and associated complement activation would alter the serum levels of the major regulators of both C3a and C5a complement split products. These serum regulators designated chemotactic factor inactivator (CFI) and anaphylatoxin inactivator (AI) were measured in the serum of patients undergoing CPB. In these studies, we demonstrated that during CPB a rapid and dramatic drop in the anaphylatoxin inactivator activities occurred within the first 10 minutes of CPB. These lowered AI levels were maintained throughout the CPB but AI levels returned to normal within 24 hours postsurgery. CFI levels were generally maintained throughout the CPB surgery with only minimal depressions in CFI levels during or after CPB surgery. These studies clearly demonstrate that the major regulator system of the complement-derived vasopermeability factors (C3a and C5a) is dramatically depressed during cardiopulmonary bypass and may suggest that the mechanisms of interstitial edema associated with pump lung syndrome may at least, in part, be related to the loss of the serum regulator enzyme carboxypeptidase N, also designated AI.

Elevation of serum chemotactic factor inactivator activity in rabbits induced by inflammation and chemotactic factor.

Inflammatory mediators such as the vasoactive and chemotactic factors, which are formed during the activation of serum complement, are extremely potent biologic peptides and are thought to be under the rigid control of specific serum-derived regulators or inactivators, including the anaphylatoxin inactivator (AI) and the chemotactic factor inactivator (CFI). Our understanding of the biologic importance and implication of these regulators in inflammatory reactions is primarily based on in vitro observations, and there is only limited data on their in vivo importance. Previously, alterations in CFI and AI levels were detected in chronic inflammatory disease states, but no data on their activity or role during acute inflammatory reactions have been demonstrated. Here we demonstrate the elevation of serum CFI activity during acute inflammatory reactions in rabbits. Specifically, inflammatory reactions were induced in rabbits by intraperitoneal injections of 0.1% oyster glycogen. Rabbit serum CFI levels rose rapidly over the first 4 hours after glycogen injection, reaching levels 4-8 times higher than the preglycogen serum CFI levels. Additional studies demonstrated that acute elevations of serum CFI levels could also be induced in rabbits by intravenous infusion of activated rabbit serum or C5-derived chemotactic factors. Infusion of saline, albumin, or the synthetic chemotactic peptide f-Met-Leu-Phe did not cause elevation of the serum CFI levels in rabbits. Thus, we take these data to support our hypothesis that 1) CFI is a "hyperacute phase reactant" that is elevated during inflammatory reactions, and 2) that this elevation of serum CFI activity is probably triggered by the appearance of specific C5-derived chemotactic factors within the vasculature. These studies not only provide exciting new insights into the regulatory mechanisms involved in acute inflammatory reactions but suggest that novel approaches to antiinflammatory therapy may be forthcoming.

Alterations in activities of anaphylatoxin inactivator and chemotactic factor inactivator during hemodialysis.

The systemic infusion of complement-derived anaphylatoxin ane chemotaxins during hemodialysis results in profound transient neutropenia and may be associated with subtle pulmonary dysfunction. The fact that these potent inflammatory peptides do not usually produce serious ill effects may be due in part to their rapid inactivation by the serum regulatory proteins anaphylatoxin inactivator (AI) and chemotactic factor inactivator (CFI). Accordingly, the authors investigated the effect of hemodialysis on circulating neutrophil counts and serum levels of AI and CFI activity in 10 patients. In all patients, circulating neutrophil counts plummeted by more than 50% within 5 minutes of the onset of dialysis and rose beyond control levels by 1 hour. AI activity significantly fell from 65 +/- 16 mU/ml before dialysis to 18.7 +/- 7.8 mU/ml within 5 minutes of its initiation; levels remained depressed throughout the procedure. In contrast, CFI gradually increased, achieving a level significantly different from the predialysis value by 15 minutes. These events contrasted with the observation that activation of the complement system in vitro results in a significant decline in both CFI and AI activities. We suggest that the maintenance of or increase in CFI activity in patients undergoing hemodialysis enhances the clearance of circulating chemotactins, preventing the persistent activation of neutrophils during the procedure and subsequent organ dysfunction.