Morphologic and functional characteristics of alveolar macrophages following cryopreservation.

Cryopreservation of nucleated blood and bone marrow mononuclear cells has been used both to preserve such cells for clinical, diagnostic, or research use and to eliminate them as passengers in frozen tissue destined for transplantation. Although techniques for macrophage and monocyte cryopreservation have been described, little work has been done on the functional state of these cells following frozen storage. Using rabbit alveolar macrophages (AM), we have shown that AM can be frozen, stored, and recovered without morphologic changes. Furthermore, freshly isolated and cryopreserved AM do not differ in their adherence characteristics or the organization of their actin cytoskeleton. Cryopreserved normal AM also retained inducible functions such as superoxide anion (O2-) release and production of tumor necrosis factor (TNF) and interleukin-1 (IL-1). In order to further define the effects of cryopreservation on macrophage biology, we have also frozen macrophages which have been primed in vitro, and have demonstrated that the primed state remains at prefreezing levels after thawing and subsequent analysis. These data help define the effects of cryopreservation on cell function and establish parameters for the clinical and experimental evaluation of cryopreserved mononuclear phagocytes.

Relationships between pulmonary inflammation, plasma transudation, and oxygen metabolite secretion by alveolar macrophages.

We have previously shown that alveolar macrophages from normal rabbit lungs do not synthesize reactive oxygen intermediates unless first conditioned by culture in vitro in the presence of serum for 24 to 48 hr. This conditioning process is mediated by a serum constituent that partitions on gel exclusion columns with an apparent m.w. of 30,000 to 50,000 daltons. Alveolar macrophage conditioning in vitro requires protein synthesis, is associated with the generation of membrane NADPH oxidase activity, and is reversible. We have predicted therefore that during the course of pulmonary inflammation, as observed 3 wk after i.v. injection of M. butyricum in oil, alveolar macrophages might similarly become conditioned in vivo through exposure to plasma protein transudates reaching the alveolus. In support of this hypothesis we show that after experimental production of granulomatous pulmonary inflammation in rabbits, alveolar macrophages showed an augmented capacity to secrete superoxide anion when stimulated with phorbol ester, and this enhancement increases exponentially with increased plasma transudation. This augmented enhancement was reversible, and decreased after culture in vitro in the absence of serum. Mature alveolar macrophages were responsible for this enhanced superoxide anion production rather than freshly emigrated monocytes. Moreover, superoxide anion production in this model of pulmonary inflammation appears to be an "all-or-none" phenomenon, with superoxide anion production associated with a subpopulation of optimally conditioned alveolar macrophages, whereas the remaining unconditioned alveolar macrophages produce little or none. We feel that these two classes of alveolar macrophages may be derived from inflamed and noninflamed regions of the lung, respectively, thereby reflecting the discontinuous nature of the inflammatory lesions themselves. Thus we propose that measurements of reactive oxygen intermediate production by lavaged alveolar macrophages may provide a semi-quantitative measure of chronic pulmonary inflammation.

Serum factor requirement for reactive oxygen intermediate release by rabbit alveolar macrophages.

Alveolar macrophages (AM) from pathogen-free rabbits were unable to release reactive oxygen intermediates (ROI) unless they were conditioned in serum for 24-48 h before triggering with membrane-active agents. The degree of serum conditioning of AM depended upon the concentration of serum used; optimal ROI release was obtained at or above 7.5% fetal bovine serum (FBS). FBS, autologous rabbit serum, pooled rabbit serum, and pooled human serum were each capable of conditioning AM for release of ROI. Serum conditioning of AM requires synthesis of new protein(s); and the enzyme required for ROI production, NADPH oxidase, was only detectable in serum-conditioned cells. Moreover, serum-conditioned cells lost their ability to release ROI after transfer to serum-free medium, while cells maintained in serum-free medium acquired the capacity to release ROI after their transfer to serum-containing medium, demonstrating the reversibility of the phenomenon. Initial purification data indicate that conditioning is mediated by a discrete serum constituent, which precipitates 40-80% saturated ammonium sulfate, does not bind to Cibacron Blue columns, and has a molecular weight of 30,000 to 50,000, as determined by molecular exclusion chromatography. Unlike gamma interferon, which also enhances ROI release by macrophages, our serum-conditioning factor is not acid labile, retaining 67% of its activity after 120 min incubation at pH 2.0. Moreover, it does not appear to be a contaminating endotoxin, since LPS neither conditioned AM for ROI production, nor triggered ROI production by serum-conditioned AM. We propose that such a conditioning requirement may normally protect the lung against ROI-mediated tissue injury. However, during a pulmonary inflammatory reaction initiated by other mediator systems, the resulting transudation of plasma proteins into the alveolar spaces may condition AM in situ for ROI production.

Immunologic mechanisms of parenchymal lung injury.

The lung, like most other organs, is susceptible to injury by circulating immune complexes, and also by humoral autoantibody and immune lymphocytes which specifically recognize selected lung antigens. In addition, by virtue of its direct communication with the external environment, the lung can also be injured by inhaled environmental agents which trigger inflammatory reactions mediated by immune effector systems. Although major emphasis to date has been placed on the ability of inhaled antigens to first sensitize, then provoke, immunologically specific reactions in the lung, there is increasing evidence to show that these same immune effector systems are also triggered in an immunologically nonspecific fashion by a certain environmental agents (termed "mitogens") which activate leukocytes in a polyclonal fashion. Such agents include certain viruses and other microorganisms, bacterial endotoxin, a wide variety of plant lectins, and certain chemicals, such as the phorbol esters. Although such agents act in an immunologically nonspecific fashion, they are nonetheless quite specific from a chemical viewpoint, and in many cases act by binding to specific receptors on the cell surface. By activating macrophages directly, and by activating much larger percentages of a given lymphocyte population than do specific antigens, they induce correspondingly amplified inflammatory reactions in vivo. Recent studies with animal models indicate that inhaled mitogens are strikingly effective in inducing pulmonary inflammation, whereas inhaled antigens (lacking mitogenic activity) produce little if any parenchymal injury in immunized recipients, unless administered in conjunction with a mitogen. Ongoing studies using such models promise to provide valuable new insight into the biologic properties which govern the pathogenicity of inhaled environmental agents, the mediators they release, and the biochemical basis for variations in individual susceptibility to injury by such agents.

The role of subcellular factors in pulmonary immune function: physicochemical characterization of two distinct species of lymphocyte-activating factor produced by rabbit alveolar macrophages.

T cell stimulatory factors produced by rabbit alveolar macrophages were investigated. Physicochemical characterization revealed that alveolar macrophages (harvested by bronchopulmonary lavage and stimulated in tissue culture with bacterial lipopolysaccharide) released 2 predominant species of lymphocyte-activating factor (LAF) with isoelectric points of 4.6 and 7.4, and m.w. of 14,400 and 11,600 daltons, respectively, as calculated by the Svedberg equation. Using C3H/HeJ mouse thymocytes (and in some instances nylon wool-purified nonadherent rabbit spleen or lymph node cells) as target cells, rabbit LAF was found to induce proliferative responses directly, as well as enhance proliferative responses to phytomitogens. Both LAF species were inactivated by heating, treatment with trypsin, or at low (2.3) pH. The pI 7.4 LAF was also unstable at high pH (9.0). The thymocyte stimulatory activity of both LAF species was not inhibited by the anti-proteases alpha-1-anti-trypsin, Traysylol (aprotinin), leupeptin, or pepstatin.

Endogenous pyrogens made by rabbit peritoneal exudate cells are identical with lymphocyte-activating factors made by rabbit alveolar macrophages.

Purified endogenous pyrogen (EP), isolated in one laboratory from stimulated rabbit peritoneal exudate cells (PEC), was compared with purified lymphocyte activating factor (LAF), isolated in another laboratory from stimulated rabbit alveolar macrophages (AM). Both EP and LAF occurred in two forms, one with an isoelectric point (pI) of 7.3 to 7.4, the other with a pI of 4.6 to 4.7. Both forms of EP and LAF had m.w. of 13,000 to 16,000 daltons as judged by gel filtration. Both forms of EP had LAF activity, and both forms of LAF were pyrogenic. The pI 7.3 EP, which is known to have an -SH group essential for its biologic activity, bound to Thiol Sepharose columns and could be eluted with mercaptoethanol. The pI 7.3 LAF behaved in exactly the same way. Furthermore, antisera from three different goats were available that completely blocked the pyrogenicity of pI 7.3 EP in vivo; these sera also blocked the pI 7.3 LAF activity in vitro. The pI 4.6 EP and LAF did not bind to Thiol Sepharose columns, nor were they inhibited by any of the antisera that blocked the pI 7.3 EP and LAF. Moreover, isoelectric focusing of pI 4.6 EP in very shallow gradients revealed microheterogeneity with sharp peaks of EP activity observed at pH 4.6 and 4.7. Analysis for LAF activity showed an identical microheterogeneity. These results are consistent with the idea that EP and LAF are identical.

Role of polyclonal cell activation in the initiation of immune complex-mediated pulmonary injury following antigen inhalation.

The lung, by virtue of its anatomic situation, provides environmental antigens with unique access to host lymphoid tissues. In order to better understand the biologic consequences of antigen inhalation, we developed in animal model in which soluble proteins are administered in aerosol form to rabbits. By labeling these proteins with fluorochrome dyes or radioactive isotopes, the uptake, distribution, and fate of such proteins can be demonstrated both morphologically and quantitatively. Prompt host-antibody responses can be demonstrated to inhaled antigen, but not to comparable amounts of ingested antigen. Repeated administrations of antigen aerosol to immune animals produced little injury; in contrast, administration of aerosols containing phytohemagglutinin or cancanavalin A (Con A), plant lectins which activate leucocytes in a polyclonal fashion, induced a diffuse interstitial pneumonitis. When immune animals inhaled antigen plus Con A, devastating pulmonary necrosis was induced, in association with localized deposits of immune complexes containing antigen, antibody and complement. Such necrotic injury healed by scarring within 4 weeks. The necrotizing injury could be prevented by either decomplementation with cobra venom factor, or through inhibition of leucocyte responsiveness to Con A by administration of cholera toxin, a cAMP agonist. These studies indicate that antigen inhalation may serve as an important means of establishing "natural" immunity to environmental agents, but also may lead to severe pulmonary injury and fibrosis where the agents inhaled act not only as antigens but as polyclonal leucocyte activators as well.

In vivo responses to inhaled proteins. III. Inhibition of experimental immune complex pneumonitis after suppression of peripheral blood lymphocytes.

We have previously shown that inhaled Con A has a powerful enhancing effect on the formation of immune complexes between an inhaled antigen and circulating antibody. Immunohistochemical staining has demonstrated such complexes, together with host complement, in close association with foci of necrotizing destruction of the pulmonary parenchyma. We have postulated that Con A promotes immune complex formation indirectly through polyclonal activation of lymphocytes in the lung. In this paper we test this hypothesis in animals rendered unresponsive to Con A stimulation in vivo by i.v. administration of cholera toxin (CT). Such treatment raised the levels of cAMP in peripheral blood lymphocytes and inhibited their proliferative response to Con A in vitro. CT administration further blocked the local inflammatory response to intradermal injections of Con A, as well as the cell-mediated immune response to intradermal injections of BSA. Although CT failed to block the immune complex-mediated Arthus vasculitis in the skin, it did block production of immune complex pulmonary injury by antigen/mitogen aerosols, as did decomplementation with purified cobra venom factor. These findings support the hypothesis that polyclonal activation of pulmonary lymphocytes promotes immune complex-type alveolitis, possibly by facilitating interactions between humoral antibody and intra-alveolar antigen.

In vivo responses to inhaled proteins. II. Induction of interstitial pneumonitis and enhancement of immune complex-mediated alveolitis by inhaled concanavalin A.

An animal model of environmental lung disease is described in which phytomitogen, antigen, or both, are administered in aerosol form to previously immunized or immunologically naive rabbits. Inhalation of concanavalin A alone induced an interstitial pneumonitis in nonimmunized rabbits. Inhalation of concanavalin A alone induced an interstitial pneumonitis in nonimmunized rabbits. Inhalation of bovine serum albumin (BSA) alone typically produced only focal eosinophilic granulomas in BSA-immunized animals, and no injury whatever in nonimmune animals. However, simultaneous administration of BSA-concanavalin A aerosol mixtures to BSA-immunized rabbits induced a severe interstitial pneumonitis and granulomatous vasculitis, together with areas of frank parenchymal necrosis. When repeated on a chronic basis over a 4- or 8-week interval, challenge with BSA-concanavalin A aerosols resulted in both acute necrotic lesions as well as areas of frank interstitial fibrosis. Necrotic foci in acutely injured lungs were associated with interstitial deposits of BSA, rabbit anti-BSA antibody, and complement. Electron microscopy revealed numerous neutrophils within the pulmonary interstitial spaces of these animals, often in association with collagen and elastin fibers. The pattern of injury in immune rabbits induced by antigen-concanavalin A aerosols, in its nonnecrotizing form, is consistent with that of an extrinsic allergic alveolitis. However, the severe, necrotizing form of acute injury closely resembles changes seen in Wegener's granulomatosis. Possible mechanisms of injury produced by antigen and phytomitogen inhalation are discussed.