Assessment of chemokinetic behavior of inflammatory lung macrophages in a linear under-agarose assay.

Migration of cells in response to a chemoattractant gradient is influenced by directed migration (chemotaxis) and stimulated random motility (chemokinesis). The present study quantitated the chemokinetic motility of normal and inflammatory lung macrophages by performing the linear under-agarose assay in the presence of uniform concentrations of chemoattractant. Under these conditions, cell motility can be likened to a molecular diffusion process. Mathematical analyses which describe molecular diffusion were then applied, allowing the quantitation of the parameter, mu, the cellular equivalent to the molecular diffusivity constant. Determination of changes in mu as a function of chemoattractant concentration revealed that the chemokinetic motility of alveolar macrophages recovered during the early stages of acute pulmonary inflammation was greater than that of normal alveolar macrophages and macrophages recovered later in the inflammatory response. The correlation of differences in macrophage chemokinesis with macrophage maturation and the relevance of these differences to macrophage accumulation during inflammation are discussed.

Pathogenesis of acute pulmonary inflammation in strain 2 and strain 13 guinea pigs.

An acute inflammatory response was elicited in the lungs of strain 2 and 13 guinea pigs following immunization and aerosol challenge with ovalbumin. The pulmonary inflammatory response, characterized by hemorrhage and influx of inflammatory cells, progressed from initiation at 12-hours postchallenge through resolution at 96-hours postchallenge. Inflammatory and immunoregulatory cells, recovered by bronchoalveolar lavage, showed quantitative changes in their relative contribution to the bronchoalveolar cell infiltrate over the course of inflammation. Changes in concentrations of macrophages and T cells, in particular, are discussed in terms of their possible contributions to initiation and resolution of acute pulmonary inflammation.

Cellular immunoregulation of acute pulmonary inflammation in strain 2 guinea pigs.

Subclasses of lung immunoregulatory T cells were analyzed during acute pulmonary inflammation in strain 2 guinea pigs and compared with T cell subpopulations in the peripheral circulation. Immunized animals were aerosol-challenged with specific antigen and sacrificed at 12-, 24-, 48-, 72-, and 96-hours postchallenge. Mononuclear cells, isolated from peripheral blood and bronchoalveolar lavage, were enriched for T cells. The percentage of helper T cells, as well as antigen-specific blastogenesis, in recovered pulmonary T cells exhibited maximal values at 12-, 24-, and 96-hours postchallenge. In contrast, the presence of suppressor T cells correlated with decreased blastogenesis and antigen-specific suppression in isolated lung cells at 72-hours postchallenge. Since changes in pulmonary cells did not correlate with those found in the peripheral circulation, immunoregulatory events in these two compartments may be distinct. These results indicate that the proportions of lung T cell subclasses, as well as their in vitro functional activity, are altered over the course of pulmonary disease. Such changes in immunoregulatory cell populations may be important in the mediation of disease pathogenesis.

Splenic regulation of the murine pulmonary lymph node response.

Exposure to intratracheal immunization and aerosolization with soluble antigen plus murmayl-dipeptide (MDP) induces the development of plaque-forming cells in the pulmonary draining lymph nodes of two of three inbred mouse strains. Splenectomy before immunization led to a heightened plaque-forming cell response in the two responder mouse strains. Adoptive transfer of spleen cells from one strain exposed to sperm whale myoglobin via the respiratory tract revealed the presence of antigen-specific suppressor cells. These observations suggest that the spleen may play a role in the down-regulation of an immune response elicited in the pulmonary draining lymph nodes by exposure of the respiratory tract to soluble antigens plus MDP.

Hemolytic plaque inhibition by synthetic antigenic peptides of sperm whale myoglobin.

We have investigated the systemic antibody response to sperm whale myoglobin (SWMb) antigenic sites in three strains of inbred mice using an inhibition of plaque assay. Sperm whale myoglobin was attached to sheep red blood cells (SRBC) via rabbit anti-SRBC Fab' fragments. Inhibition of lysis was obtained with synthetic peptides representing the purported five antigenic sites but not with a peptide whose sequence was unrelated to SWMb and synthetic peptides of SWMb from outside the antigenic sites gave minimal or no inhibition. The results of our studies show that the pattern of response to the five antigenic sites differs in each strain, but that almost total inhibition is obtained in all strains with these five sites. The antigenic dominance of these sites supports the concept of discrete antigenic sites on soluble proteins. They also suggest a reason for contrary reports in the literature based on hybridoma technology.

Pulmonary immune effector cells: II. Antigen-specific blastogenic responsiveness of lymphocyte populations during pulmonary immune complex disease in guinea pigs.

A guinea pig pulmonary immune complex disease was used to evaluate local antigen (ovalbumin)-specific lymphoproliferative responses in lung tissue, bronchoalveolar spaces, and hilar lymph nodes (HLN) at various time intervals after challenge. The responses of lung tissue and bronchoalveolar lymphocytes appear to be mediated by T cells, whereas the response of HLN lymphocytes was mediated by B and/or T cells, depending on the stage of the disease. The blastogenic response of HLN lymphocytes to concanavalin A was much greater than that observed in lung tissue or bronchoalveolar lymphocyte preparations, even after the removal of adherent cells, suggesting a possible inherent difference between these cell populations in their response to mitogen. This study demonstrates that lung tissue, bronchoalveolar, and HLN lymphocytes are not only capable of responding blastogenically to specific antigen, but that this responsiveness varies throughout the course of the disease. The lymphoproliferative responses and concurrent changes in the proportion of pulmonary immune effector cells are discussed in relation to cellular immunoregulation during the in vivo progression of this pulmonary immune complex disease.

Pulmonary immune effector cells in guinea pigs with immune complex disease. I. Changes in T- and B-lymphocyte populations after exposure to antigen.

Changes in immunologic effector cell populations in lung tissue, bronchoalveolar spaces, tracheobronchial lymph nodes, spleen, and peripheral blood were evaluated during the course of a pulmonary immune complex disease in guinea pigs. The number of macrophages, lymphocytes, and neutrophils present in each cell population were determined. T and B lymphocytes were identified by E and EAC rosette formation, respectively. An increase in the total number of lymphocytes in tracheobronchial lymph nodes and a greater proportion of B cells in these lymphocyte populations were observed at 12 and 24 hr postchallenge. The total number of macrophages, lymphocytes, and neutrophils recovered from the bronchoalveolar spaces also increased, as did the proportion of lymphocytes and neutrophils. A similar proportional increase of lymphocytes obtained from lung tissue also occurred. The proportion of B cells in the lymphocyte populations of the bronchoalveolar spaces and lung tissue increased to a maximum at 24-48 hr postchallenge. Cell populations from peripheral blood or spleen remained stable, by all parameters examined, during the disease process. Thus, there appears to be a localization of the immune inflammatory response in the lungs during the course of this pulmonary immune complex disease. In addition, this study provides evidence that immune effector cells obtained by bronchial lavage accurately reflect the cellular changes associated with the acute inflammatory response in lung tissue and pulmonary lymph nodes.

Immune complex disease in guinea pig lungs: elicitation with pigeon serum.

Immune complex- and T cell-mediated reactions to organic antigens appear to contribute to the pathogenesis of hypersensitivity pneumonitis in humans. Because pigeon serum is one of the reagents used by clinicians to diagnose this disease, we assessed its potential to elicit immune complex-mediated pulmonary inflammation in guinea pigs. Animals were immunized with different concentrations of pigeon serum protein emulsified in complete Freund's adjuvant, and serums were collected at 4-day intervals after the booster injection. The largest amounts of tissue-fixing (IgG1) and complement-fixing (IgG2) antibodies to pigeon serum were detected in guinea pigs immunized with 1.0 mg of pigeon serum protein 20 to 24 days after the secondary immunization. Therefore, the responses of these animals and of recipients of serum from these animals to aerosol challenge with either homologous (pigeon serum) or heterologous (bovine gamma globulin) immunogen was investigated. Actively and passively immunized guinea pigs developed pulmonary inflammation only after exposure to aerosolized pigeon serum. However, lesions were not observed in the lungs of complement-deficient recipients of immune serum that had inhaled homologous immunogen. These observations suggest that such pigeon serum-elicited pulmonary inflammation in guinea pigs is a manifestation of a complement-dependent, humoral-immune mechanism of pathogenesis and thus is consistent with an immune complex disease.

Immunologically induced lung disease in guinea pigs. A comparison of ovalbumin and pigeon serum as antigens.

This study was conducted to compare the capacity of pigeon serum (PS), an antigen (Ag) associated with hypersensitivity pneumonitis (HP), and ovalbumin (OA) in the induction of immunologic lung disease in guinea pigs (gp). Whereas OA was very effective in inducing a severe pneumonitis, PS failed to produce significant disease. A determination of the antibody (Ab) responses in OA- or PS-sensitized GP revealed that total Ab activity, as well as specific IgG1, and IgG2 responses, were not significantly different in the two groups. There was, however, a markedly higher IgE-like Ab response to OA than to PS. Thus, there was a striking correlation between specific IgE synthesis and the production of immunologic lung disease. The disease resembled immune complex disease histologically, and we suggest that the IgE antibody may function as a preceding "anaphylactic trigger" mechanism for the lodging of complement-fixing Ag-Ab complexes in the vasculature of the lung. It is further suggested that PS may be a poor Ag for the induction of IgE synthesis in guinea pigs.