Effect of bronchoalveolar lavage fluid from Pneumocystis carinii-infected hosts on phagocytic activity of alveolar macrophages

Abstract

Alveolar macrophages from Pneumocystis carinii-infected rats are defective in phagocytosis. To investigate whether this defect is due to a certain factor present in P. carinii-infected lungs, alveolar macrophages from uninfected rats were incubated with bronchoalveolar lavage (BAL) fluid samples from P. carinii-infected rats. Alveolar macrophages treated with these BAL fluid samples became defective in phagocytosis but remained normal when treated with BAL fluid samples from noninfected or Toxoplasma gondii-infected rats. The suppressive activity of the BAL fluid samples from P. carinii-infected rats on phagocytosis was retained when the BAL fluid samples were passed through a filter with a pore size of 0.45 microm but was lost when the BAL fluid samples were digested with proteases such as trypsin, pepsin, papain, or endopeptidase Gly-C. Lipid fractions of these BAL fluid samples had no suppressive activity on phagocytosis. The suppressive activity of these BAL fluid samples was also lost when they were incubated with concanavalin A-agarose beads, suggesting that the inhibitor is a glycoprotein. The inhibitor was estimated to be larger than 100,000 Da by exclusion filtration. After binding to the concanavalin A-agarose beads, the inhibitor in BAL fluid samples and P. carinii lysate could be eluted with 200 mM methylmannose. Treatment of both the crude BAL fluid samples and P. carinii lysate and the 200 mM methylmannose eluate with antibody against the major surface glycoprotein of P. carinii eliminated their suppressive activity. These results suggest that the factor capable of suppressing the phagocytic activity of alveolar macrophages is P. carinii major surface glycoprotein or one or more of its derivatives.

FIG. 1.

Phagocytosis of latex beads by alveolar macrophages from T. gondii-infected rats. Immunosuppressed rats were transtracheally inoculated with 100 (Toxoplasma-100) or 10 (Toxoplasma-10) T. gondii tachyzoites. Three weeks after initiation of infection, alveolar macrophages and BAL fluid were recovered. (A) Alveolar macrophages from T. gondii-infected rats were able to phagocytose latex beads as efficiently as those from uninfected controls (dexamethasone treated). T. gondii burdens were higher in the animals that received 100 tachyzoites (P < 0.05), but this increased burden did not affect phagocytosis ability (P > 0.05 versus 10 tachyzoites). Open columns indicate levels of phagocytosis, and shaded columns indicate T. gondii organism burdens. (B) Alveolar macrophages from normal and dexamethasone-treated rats were incubated with 500 μl of PBS (open columns) or BAL fluid (shaded columns) (500 μl at 1 mg of protein per ml) from the most heavily T. gondii-infected rats from the experiments in panel A for 12 h and then assessed for the ability to phagocytose latex beads. Phagocytosis of latex beads by alveolar macrophages from normal and Dex rats was unaffected by incubation with BAL fluid samples from T. gondii-infected rats (P > 0.05).

FIG. 2.

Effect of BAL fluid samples from normal, Dex, and Dex-Pc rats on phagocytosis by alveolar macrophages. (A) Alveolar macrophages from normal, Dex, and Dex-Pc rats were incubated with BAL fluid samples (0.5 mg of protein) from moderately (lightly shaded columns) or severely (darkly shaded columns) infected Dex-Pc rats. Compared to the PBS control (open columns), BAL fluid from both moderately and severely infected rats was able to inhibit phagocytosis of latex beads by alveolar macrophages from both normal and Dex rats (P < 0.05). (B) BAL fluid samples from normal and Dex rats were unable to restore the phagocytic activity of alveolar macrophages from Dex-Pc rats (P > 0.05). Alveolar macrophages from Dex-Pc rats were unable to phagocytose increased numbers of latex beads despite having been removed from the infected host for 24 h and incubated with BAL fluid samples from uninfected rats for 12 h.

FIG. 3.

Effect of filtered BAL fluid on phagocytosis of latex beads by alveolar macrophages. Normal and Dex rats were lavaged for alveolar macrophages, and these cells were incubated for 12 h with BAL fluid (0.5 mg of protein) that had been filtered through a 0.45-μm filter. The alveolar macrophages were then assessed for their ability to phagocytose 1-μm-diameter latex beads. Results are averages ± standard deviations from triplicate phagocytosis assays performed for each of five experiments for each condition. For alveolar macrophages from both normal and Dex rats, incubation with Pc-BAL fluid samples (darkly shaded columns) significantly reduced phagocytosis (P < 0.05 versus normal BAL fluid samples), while BAL fluid samples (lightly shaded columns) from immunosuppressed rats had no effect on phagocytosis (P > 0.05 versus normal BAL fluid samples).