The effect of pulmonary surface-active material on the generation and expression of murine B- and T-lymphocyte effector functions in vitro.

We demonstrated previously that surface-active material potently suppresses early proliferative responses of lymphocytes to a wide variety of immune stimuli in vitro. It is now evident that in vivo, effector B and T lymphocytes can be recruited into lung parenchyma subsequent to their generation in extrapulmonary lymphoid tissues. The purpose of the present study was to examine the effects of surface-active material on proliferation, differentiation, and expression of effector functions of cytotoxic T cells and antibody-forming B cells in vitro in order to gain insight into the potential immune regulatory role of surface-active material in vivo. Normal spleen lymphocytes were cultured in vitro for 5 days with either allogeneic lymphocytes to generate cytotoxic T cells or with sheep erythrocytes to generate antibody-forming B cells. Surface-active material was added at various intervals after the cultures were initiated, and the effects of such additions on the subsequent proliferation, differentiation, and expression of cytotoxic T cells and antibody-forming cells were determined. Addition of surface-active material on days 0 through 3 suppressed both lymphocyte proliferation and the subsequent differentiation of effector lymphocytes. By contrast, addition of surface-active material after day 3 exerted no measurable effect on proliferation or on the generation of effector lymphocytes. We conclude that in vitro the immunosuppressive activity of surface-active material is exerted primarily during early proliferative phases of immune responses and that once these have occurred, surface-active material does not inhibit the later stages of differentiation and expression of effector cell functions. We speculate that in vivo, surface-active material may suppress local proliferation of lymphocytes resident in the lung in response to inhaled antigens; however, it may not interfere with effector functions of partially or fully differentiated B and T lymphocytes that are recruited into lungs from systemic sources.

Procoagulant activity of rabbit alveolar macrophages.

Alveolar macrophages are thought to participate in the clearance of fibrin from the injured lung, but their ability to facilitate the conversion of fibrinogen to fibrin (procoagulant activity) has not been described. In order to characterize their procoagulant properties, unstimulated alveolar macrophages obtained from normal rabbits were tested for their ability to accelerate the coagulation of plasma in a one-stage clotting assay. Compared with control assays containing no macrophages (coagulation times greater than 500 s), intact cells (10(6)/ml) were shown to display procoagulant activity (coagulation time, 153.6 +/- 11.3 s mean +/- SEM). Cell lysis caused further procoagulant activity to be expressed (125.6 +/- 11.8 s). Alveolar macrophages that were stimulated in vitro with bacterial lipopolysaccharide (LPS) or the purified complement fragments C5a and C5a des Arg caused further significant (p less than 0.002) reductions in coagulation times (intact cells, 71 to 76 s; lysed cells, 27 to 32 s), representing 5- to 6-fold and 30- to 40-fold increases in the procoagulant activity of intact and lysed cells, respectively. The generation of this material was independent of the presence of lymphocytes. The procoagulant material was identified as a cell-associated tissue thromboplastin, acting via the extrinsic coagulation pathway. These findings show that alveolar macrophages have procoagulant activity that is markedly augmented by LPS and complement fragments. This suggests that alveolar macrophages may contribute to intra-alveolar fibrin deposition in vivo.

Hyporesponsiveness of canine bronchoalveolar lymphocytes to mitogens: II. Analysis of macromolecular synthesis, T-cell antigen, and mitogen-binding.

Canine bronchoalveolar lymphocytes (BAL) and peripheral blood lymphocytes (PBL) were prepared by filtration over nylon wool columns and were studied for their ability to: (a) synthesize protein, RNA, and DNA after mitogen stimulation; (b) express T-cell antigen; and (c) bind radiolabeled mitogen. Bronchoalveolar lymphocytes were shown to be markedly hyporesponsive to mitogen stimulation as assessed by DNA synthesis after 72 hr of culture or by protein and RNA synthesis after 24 hr of culture. The hyporesponsiveness of BAL was not due to a decrease in the number of T-cells; results of an indirect immunofluorescent assay employing a rabbit antiserum indicated similar percentages of T-cells in BAL (78%) and PBL (79%). The hyporesponsiveness was not due to abnormalities in mitogen binding by BAL; results of a radiolabeled mitogen-binding assay indicated essentially no difference in mitogen receptor number or affinity between BAL and PBL. Thus, the initial interaction bronchoalveolar T lymphocytes and mitogen appears to be normal. This normal interaction with mitogen, however, fails to trigger an appropriate level of macromolecular synthesis by BAL. This suggests that the hyporesponsiveness of BAL is due to an abnormality in one of the early metabolic processes induced by the interaction of a mitogen with the cell membrane.

Identification of the immunosuppressive components of canine pulmonary surface active material.

Pulmonary surface active material (SAM), purified from canine lung lavage fluids, is a phospholipid-rich lipoprotein with potent immunosuppressive activity. Experiments were performed to identify those components of SAM that were responsible for this immunosuppressive effect. Results indicated that the lipid, and not the protein, fraction of SAM was immunosuppressive. Two phospholipids, phosphatidylglycerol and phosphatidylcholine, were identified as the predominant immunosuppressive components of the SAM-lipid fraction. lymphocyte proliferation in response to mitogenic or allogeneic stimulation was suppressed by intact SAM, SAM-lipid, phosphatidylglycerol, and phosphatidylcholine. Each of these preparations inhibited RNA, protein and DNA synthesis by mitogen-stimulated lymphocytes. Antioxidants consistently failed to diminish the immunosuppressive properties of SAM or its lipid components. The mechanism of this immunosuppressive action of SAM and its phospholipid components remains undefined. Our data indicate, however, that it is unlikely to be due either to cytotoxicity or to an artifact of lipid oxidation in vitro.

Canine surface active material and pulmonary lymphocyte function. Studies with mixed-lymphocyte culture.

Canine bronchoalveolar cells, obtained by lavage, were enriched for lymphocytes by adsorption to plastic or by filtration over nylon wool and tested for their ability to function in the mixed lymphocyte culture (MLC) reaction. Pulmonary lymphocytes were markedly hyporesponsive to stimulation with allogeneic cells in vitro: their responses rarely exceeded 10% of those of blood lymphocytes obtained simultaneously from the same donor. However, pulmonary lymphocytes did function as stimulating cells, inducing allogeneic blood lymphocytes to proliferate in MLC. The failure of pulmonary lymphocytes to respond in MLC, coupled with their ability to stimulate clearly, distinguishes these cells from circulating blood lymphocytes. The effect of canine surface active material (SAM), a lipoprotein unique to the lung, on the function of blood lymphocytes in MLC was studied. A transient exposure to SAM in vitro profoundly suppressed blood lymphocyte responses to allogenic stimulation, but had only a minor effect on their function as stimulator cells in MLC. Thus, exposure to SAM in vitro converts normal blood lymphocytes into cells whose function mimics that of pulmonary lymphocytes. These results suggest that exposure of pulmonary lymphocytes to SAM in vivo may contribute to their abnormal immune reactivity in vitro.

An indirect assay for canine leukocyte migration inhibitory factor (LIF): demonstration of its species specificity.

An indirect assay in agarose-gel was developed to measure secretion of leukocyte migration inhibitory factor (LIF) by canine lymphocytes. Supernatants from cultures of mitogen- or alloantigen-stimulated canine peripheral blood lymphocytes were assayed for LIF activity using purified granulocytes as indicator cells. Canine LIF activity could be detected provided either autologous or allogeneic canine granulocytes were used as indicator cells. In contrast, canine LIF uniformly failed to inhibit the migration of human granulocytes. Likewise, human LIF inhibited the migration of human but not canine granulocytes in this assay. The data indicated that this species specificity could be due to differences in the structures of LIF receptors on canine and human granulocytes.

Immunosuppression by surface-active material: lack of species specificity.

Surface-active material isolated from the lungs of both dogs and rats was tested for its ability to suppress the in vitro proliferative responses of dog, mouse, or human lymphocytes to a variety of immunologic stimuli. Both dog and rat surface-active material exerted a dose-dependent suppressive effect on the proliferative responses of each species of lymphocyte, regardless of the nature of the immune stimulus (mitogen, antigen, or alloantigen). The data indicated that surface-active material acts by directly inhibiting the responding lymphocyte and not by activating suppessor cells. The immunosuppression could not be attributed to lymphocyte cytotoxicity. Although the mechanism of this immunosuppressive action of surface-active material remains undefined, the present data clearly indicate that such activity is not species specific.

Lung bacterial clearance in murine pneumococcal pneumonia.

We studied the bactericidal capacity of the rat lung during the development of pneumococcal pneumonia. Pneumonia was produced in a lower lobe by the intrabronchial instillation of 10(4)Streptococcus pneumoniae cells in buffer. Lung bacterial counts progressively increased, reaching 10(7) per lung within 48 h, and the increase was associated with localized atelectasis and consolidation. Bacterial multiplication was inhibited with tetracycline at various intervals after infection, and the subsequent clearance of pneumococci was determined. Viable pneumococci were rapidly killed by lung defenses if bacterial multiplication was inhibited within 12 h of the onset of infection. No change occurred in the bacterial populationif tetracycline was delayed until 24 h after infection, indicating that pneumococcal killing by lung defenses had ceased. This effect could be reproduced with the addition of pneumococcal capsular polysaccharide to the inoculum, which produced a dose-related inhibition of pneumococcal clearance. The clearance of S. epidermidis was not impaired in the presence of pneumococcal pneumonia or by administration of exogenous capsular polysaccharide. These data indicate that pneumococcal pneumonia causes a marked impairment in lung antipneumococcal defenses within 24 h of the onset of infection. This acquired defect in antibacterial defenses may be due to the accumulation of pneumococcal capsular material in the lungs of infected animals.