Expression of Ia by murine alveolar macrophages is upregulated during the evolution of a specific immune response in pulmonary parenchyma

These studies were performed to test the hypothesis that the evolution of a specific immune response in lung parenchyma upregulates the expression of Ia on surface membranes of murine alveolar macrophages. A secondary antibody-forming cell response to sheep erythrocytes was generated in lung parenchyma by intratracheal antigen challenge of systemically primed mice. During the immune response, alveolar macrophages were retrieved by bronchoalveolar lavage, and the percentages and total numbers of Ia-positive macrophages were measured by indirect immunofluorescence. The expression of Ia on surface membranes of lavaged alveolar macrophages increased in association with the generation of antibody-forming cell responses in lung tissue. This increase in Ia expression was antigen specific; intratracheal challenge with noncrossreacting antigen did not increase Ia expression. Nonspecific inflammation of the lung, induced by intratracheal hydrochloric acid, elicited increases in total numbers of macrophages that were similar in magnitude to those induced by specific immune responses, but increased Ia expression only modestly. In unprimed mice, intratracheal antigen challenge did not increase Ia expression by alveolar macrophages unless the mice had received immune splenocytes by adoptive transfer at the time of challenge. The results show that the generation of a specific immune response in pulmonary parenchyma upregulates the expression of Ia by murine alveolar macrophages in vivo and suggest that the accumulation of antigen-reactive lymphocytes in the lung plays an important role in this upregulation.     

The mechanism of appearance of specific antibody-forming cells in lungs of inbred mice after immunization with sheep erythrocytes intratracheally. II. Dose-dependence and kinetics of appearance of antibody-forming cells in hilar lymph nodes and lungs of unprimed and primed mice

We are conducting studies designed to define the cellular basis for the appearance and accumulation of specific antibody-forming cells (AFC) in lung parenchyma of mice after intrapulmonary deposition of sheep erythrocytes (SRBC). This study was performed: to compare qualitatively the AFC responses to intratracheally administered antigen among unprimed mice and among mice primed either by adoptive transfer of sensitized lymphocytes or by systemic immunization, and to define quantitatively the relationship between the appearance of AFC in hilar lymph nodes (HLN) and in lung parenchyma in these 3 groups of mice. Both antigen dose-response and kinetic analyses of the appearance of AFC in the HLN and lung parenchyma were performed. There was a direct relationship between the dose of SRBC administered intratracheally and the magnitude of the AFC-responses in HLN and lungs; AFC appeared in HLN at substantially lower intratracheally administered doses of SRBC than in lungs. The major effect of adoptive transfer or of systemic priming was to shift the dose-response curves significantly to the left, such that AFC appeared in HLN and lungs at lower antigen doses than in unprimed mice. Kinetic analyses showed that the initial appearance and the peak concentrations of AFC occurred earlier in HLN than they did in lung parenchyma. Priming shifted the kinetic curves to the left, accelerating the appearance of AFC; the sequential relationship between AFC appearance in HLN and in lungs was preserved.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of appearance of specific antibody-forming cells in lungs of inbred mice after intratracheal immunization with sheep erythrocytes

Immunization, ablation, and adoptive transfer studies were performed in inbred mice to define in vivo the cellular mechanisms for the appearance of specific antibody-forming cells (AFC) in pulmonary parenchyma. Mice were immunized locally or systemically with sheep erythrocytes (SRBC), and the concentrations of IgM- and IgG-producing AFC were measured in lung and extrapulmonary lymphoid tissues with a hemolytic-plaque assay. Splenectomized mice and recipients of adoptively transferred, sensitized lymphocytes were examined. We found that primary intratracheal (IT) immunization regularly failed to induce the appearance of AFC in lungs, whereas IT boosting of primed animals consistently succeeded. Immunization experiments showed that mice could be primed by any of a variety of local or systemic routes, but that the IT route of boosting was an absolute requirement for the induction of pulmonary AFC in primed mice. Recruitment of AFC into lungs by IT boosting of systemically primed and boosted animals was antigen-specific. Splenectomy performed prior to priming reduced, but did not ablate, the pulmonary AFC-response to IT boosting. Adoptive transfer of sensitized lymphocytes to naive recipient mice substituted for antigen-priming, which is required for induction of pulmonary AFC by IT challenge. Results of adoptive transfer studies demonstrate that IT challenge with specific antigen recruits systemically administered sensitized lymphocytes into the lung. We conclude that local primary immunization of mice results in the generation of AFC in extrapulmonary lymphoid tissues and that the major mechanism for the appearance of AFC in lungs is through recruitment of sensitized cells from systemic sources by intrapulmonary boosting with specific antigen.     

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.     

The effect of antigen priming on immunological response regulation in aging.

The primary anti-sheep red blood cells response and the response of antigen primed CBA mice aged 3–29 months were compared. There was a 30-fold decline with age in the primary immunological response, tested by the direct plaque-forming cell number. In young and young adult mice, priming resulted in a fall of antibody production and of number of Ig-positve spleen lymphocytes. During the lifespan the response of primed mice increased, reaching and then exceeding the level of primary response in senescence as much as 20-fold. Negative correlation was established between antibody-forming and nuclear spleen cell numbers of old primed mice.The adoptive transfer of spleen cells of primed young adult mice suppressed the antibody production in immunized recipients, whereas that of old primed spleen cells produced no reaction, the response of the recipients of old primed spleen cells being thus higher.The addition of young bone marrow to old primed mice enhanced the response and abolished the negative correlation between plaque-forming cell and nuclear spleen cell numbers.The data obtained showed a decreased suppressor activity of the antigen primed spleen cells with aging. This phenomenon seems to reflect certain features of the immune response regulation in old age.     

Cytomegalovirus-induced reactivation of Toxoplasma gondii pneumonia in mice: lung lymphocyte phenotypes and suppressor function.

Active cytomegalovirus (CMV) infection is associated with immunosuppression and predisposes to the development of life-threatening superinfections in immunocompromised patients. In a mouse model of virus-induced immunosuppression, acute murine CMV (MCMV) infection induced reactivation of dormant Toxoplasma gondii infection, producing Toxoplasma pneumonia. Changes in lung lymphocyte numbers and phenotypes appeared to be integral to the pathogenesis of MCMV-induced reactivation of T. gondii pneumonia. Numbers of lung CD4+ cells decreased during acute MCMV infection in mice with dormant T. gondii infection as well as in previously uninfected mice. Dually infected mice subsequently developed reactivation of Toxoplasma pneumonia. The pneumonia was characterized by a large influx of T lymphocytes, predominantly CD8+ cells, into the lungs. These lung lymphocytes markedly suppressed the ability of immune splenocytes to proliferate in response to T. gondii antigens and concanavalin A in vitro. These results suggested that the initial fall in the numbers of lung CD4+ cells observed after MCMV infection may have induced reactivation of T. gondii infection in the lungs. The subsequent pneumonia appeared to be a manifestation of a massive influx of T lymphocytes, especially CD8+ cells, into the lungs.     

Mouse hypersensitivity pneumonitis: depletion of NK cells abrogates the spontaneous regression phase and leads to massive fibrosis.

The contribution of natural killer cells (NK) in the progression of mouse hypersensitivity pneumonitis induced by repeated intranasal instillations with the thermophilic actinomycete Faeni rectivirgula was examined. These instillations determined a very large increase in the lung index (ca. twofold at 3 weeks), used as a measure of inflammation. In addition, this instillation was associated with a tenfold increase in the number of cells recovered in the bronchoalveolar lavage (BAL) at 3 weeks and thereafter. Most of these cells were macrophages, whereas T lymphocyte numbers increased at 6 weeks and thereafter. The instillations were also associated with a substantial fibrotic response in the lungs, as seen by large increases in hydroxyproline levels in the lungs. This fibrotic response, however, diminished after 6 weeks of instillations. Similarly, examination of histological preparations of lungs of challenged mice showed that F. rectivirgula induced inflammatory infiltrates of macrophages, lymphocytes, and neutrophils. The severity of the lesions were reduced in mice given more than 6 weeks of the actinomycete challenge. The involvement of NK cells on the development of this pulmonary pathology was determined by infusing F. rectivirgula-challenged mice with anti-NK 1.1 antibody. The depletion protocol was validated by verifying that such treatments effectively blocked lung NK cell activity. Such NK cell-depleted mice responded to the F. rectivirgula challenge with an increased lung index at 9 and 12 weeks, compared to mice challenged with F. rectivirgula and given control antibody. NK cell-depleted mice also responded to the actinomycete with a superior cellular recruitment in the BAL, with this increase mostly mediated by macrophages. Similarly, NK cell-depleted mice developed a fibrotic response that was much higher than that seen in control challenged mice, at 6, 9, and 12 weeks after initiation of the transnasal instillations. This was corroborated by scoring the severity of the histopathological lesions, which showed that NK cell-depleted mice had more severe lesions than challenged control mice. The importance of NK cells was confirmed by demonstrating that mice given anti-NK 1.1, challenged with F. rectivirgula and reconstituted with Percoll gradient-enriched lung NK cells had hydroxyproline levels at 9 and 12 weeks that were comparable to that seen in intact mice, as well as a histopathological score similar to control challenged mice. Overall, this suggests that in the course of a pulmonary inflammatory response, NK cells exert a suppressive effect on cellular recruitment in the BAL, contribute to down-regulating the inflammatory response, and are involved in blocking the appearance of fibrosis.     

Alteration of bronchoalveolar cells during murine cytomegalovirus interstitial pneumonitis

In BALB/c mice, murine cytomegalovirus (MCMV) in conjunction with a single dose of cyclophosphamide (CP) induces a diffuse interstitial pneumonitis not seen with either virus or CP alone. To gain insight into the host immune mechanisms operating in the lung during interstitial pneumonitis, we examined the cells recovered in bronchoalveolar lavage (BAL) fluids of mice with MCMV with and without CP. During MCMV interstitial pneumonitis, there was a significant increase in the total BAL cells recovered, primarily because of an influx of lymphocytes bearing the Thy 1.2 marker. Although the number of cells with Lyt 1 and Lyt 2 markers increased, the most significant increase was in the proportion of lymphocytes with surface asialo-GM1. Delineating the roles of these various cell populations may provide insight into the pathogenetic mechanisms leading to CMV interstitial pneumonitis.     

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.     

Effect of murine gamma interferon on the cellular responses to bleomycin in mice

Because in vitro studies have shown inhibition of fibroblast proliferation and collagen synthesis by interferon, we tested the hypothesis that murine gamma interferon inhibits bleomycin-induced pulmonary fibrosis in mice. Mice were divided into the following groups: saline plus vehicle (27), saline plus interferon (29), bleomycin plus vehicle (26), and bleomycin plus interferon (26). Bleomycin or saline were given intratracheally once at the beginning of the experiment and vehicle (phosphate-buffered saline) or interferon was given intramuscularly daily. Mice were killed at 14 or 21 days of the experiment. About half of the mice from each group were used for collagen biochemistry and half for bronchoalveolar lung lavage, transmission electron microscopy (TEM), and morphometry. Hydroxyproline content showed a significant reduction in bleomycin plus interferon compared to bleomycin plus vehicle mice at 21 days. The saline plus vehicle and saline plus interferon mice showed no difference in hydroxyproline content. Similarly, bronchoalveolar lavage showed no differences between saline plus vehicle and saline plus interferon mice; however, all mice treated with bleomycin showed significant increases in total cells as compared to saline treated mice. At 14 and 21 days in bronchoalveolar lavage there were significantly more lymphocytes in bleomycin plus interferon compared to bleomycin plus vehicle mice. In bronchoalveolar lavage, there were usually fewer neutrophils, monocytes and macrophages in bleomycin plus interferon compared to bleomycin plus vehicle mice. Morphometric estimates of the volume of lesion within lung showed no significant differences among the bleomycin treated groups. Stainable collagen fibers were less, but not significantly, in the bleomycin plus interferon compared to bleomycin plus vehicle mice. The number of fibroblasts per volume of lesion was significantly decreased at 14 and 21 days in bleomycin plus interferon compared to bleomycin plus vehicle mice. The total volume of lymphocytes in interstitial lesions was significantly greater at 14 and 21 days in bleomycin plus interferon mice compared to bleomycin plus vehicle mice. These results suggest an inhibitory action of gamma interferon on collagen accumulation and fibroblast proliferation associated with lymphocyte accumulation in the lungs of mice following bleomycin administration.     

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.     

Pulmonary interstitial fibrosis induced in hapten-immune hamsters

A model for pulmonary interstitial fibrosis (PIF) based on cell-mediated immune response is described. Animals were primed for contact hypersensitivity responses by skin painting with trinitrophenol (TNP), but instead of challenging with the antigen on the skin, animals were challenged with a single intratracheally administered dose of the immunizing hapten. Primed animals developed inflammation followed by pulmonary fibrosis, as determined by histologic examination. Furthermore, immunized animals developed an increase in hydroxyproline (as an indirect measure of collagen synthesis) that could be recovered from the lung by 7 days after an intratracheal challenge with TNP. The increase in hydroxyproline within the lung persisted through 30 days. The response was specific because little or no fibrosis or increase in collagen deposition was observed in immune animals that were challenged with an unrelated hapten (dinitrophenol). Unimmunized animals demonstrated a slight increase in hydroxyproline in the lung 7 days after challenge, but with time the collagen content of these control animals approached normal levels. These studies demonstrate that a specific cell-mediated immune response to a hapten within the lung can induce pulmonary interstitial fibrosis.     

Lymphocyte phenotypes in bronchoalveolar lavage and lung tissue in sarcoidosis and idiopathic pulmonary fibrosis

To determine whether the lymphocytes recovered by bronchoalveolar lavage (BAL) are representative of the cells found in lung tissue, we identified and enumerated lymphocyte phenotypes directly in lung tissue and lavage fluid with monoclonal antibodies (Leu 4-total T, Leu 3-helper T, Leu 2-suppressor/cytotoxic T cells) and an avidin-biotin peroxidase method in 6 patients with sarcoidosis and 6 patients with idiopathic pulmonary fibrosis (IPF). We found that the absolute numbers of each phenotype and the ratios of Leu 3/Leu 4, Leu 2/Leu 4, and Leu 3/Leu 2 in lavage fluid and tissue correlated well for both groups of patients. This supports the notion that the lymphocytes recovered by BAL are representative of the cells present in the lung. However, when cell recovery was expressed as the number per milliliter of recovered lavage fluid, there were no significant correlations between lavage fluid and tissue for any phenotype in the IPF group. The degree of restrictive impairment was significantly greater and the lavage fluid recovery was significantly lower in this group than in the sarcoid group. Thus, the lymphocytes recovered by BAL appear to mirror the types of cells found in lung tissue in these 2 forms of diffuse interstitial disease, but this relationship may not hold when there is a severe restrictive impairment and a low recovery of lavage fluid.     

Conjugates of ovalbumin and monomethoxypolyethylene glycol abolish late allergic responses and decrease IL-4 and IL-5 mRNA expression in the rat

The purpose of this study was to test the therapeutic potential of monomethoxypolyethylene glycol (mPEG) conjugated-allergen using a rodent model of allergic asthma. Previously, this conjugate has been shown to possess the dual capacity of inducing long-term ovalbumin (OA)-specific suppression of the antibody response and inactivating rat mast cells that have been sensitized with murine IgE to OA. Ovalbumin sensitized and challenged Brown Norway rats were studied. Fourteen days after sensitization, a test group of six rats received mPEG-OA solution intratracheally and were challenged 30 min later with aerosolized OA. Another group of seven sensitized rats was similarly challenged with OA 30 min after intratracheal administration of normal saline. A group of six sensitized rats received mPEG-OA solution intratracheally but were challenged with normal saline. Another group of seven sensitized rats received mPEG-BSA solution intratracheally and were challenged 30 min later with aerosolized OA. A final group of five unsensitized rats were neither challenged nor medicated intratracheally. Pulmonary resistance was measured before and for 8 h following inhalation challenge. mPEG-OA treatment had an inhibitory effect on the allergic late airway response, but the early response was not significantly altered. Both mPEG-OA and mPEG-BSA reduced the total cells, eosinophils and neutrophils, in bronchoalveolar lavage and decreased the expression of IL-4, IL-5 and IFN-gamma mRNA. In conclusion, mPEG-OA can prevent the development of allergen-induced late airway responses and reduce airway Th2-type cytokine expression whereas mPEG conjugated to an irrelevant antigen (BSA) is anti-inflammatory but does not affect the late response.     

致敏小鼠抗原激发后肺局部免疫反应的研究

目的：探讨抗原致敏和激发后肺局部免疫反应的变化。方法：以抗原钥孔嘁血兰素（KLH）经气管内滴注致敏小鼠，2－4周后进行激发，期间分别收集支气管肺泡灌洗液（BALF）和外周血，对肺、肺相关淋巴结（LALN）和脾组织细胞悬液进行培养并收集上清液，以酶联免疫吸附试验（ELISA）测定总IgA、抗KLHIgA及白蛋白。结果：KLH致敏和激发均引起BALF中抗KLHIgA反应。抗原致敏和激发后BALF中抗KLHIgA／白蛋白比率明显高于血清；抗原致敏激发后肺和LALN细胞悬液在体外均可释放抗KLHIgA，而脾细胞悬液仅释放低水平抗KLHIgA。结论：气管内滴注抗原致敏小鼠可诱导肺局部抗原特异IgA反应，抗原激发后反应加强，肺局部积聚的抗KLHIgA并非由血中渗漏而来，而是局部产生的结果，肺和LALN中的淋巴细胞是抗原特异IgA的主要来源。     

Requirement for leukotriene B4 receptor 1 in allergen-induced airway hyperresponsiveness

RATIONALE: Leukotriene B4 (LTB4) is a rapidly synthesized, early leukocyte chemoattractant that signals via its cell surface receptor, leukotriene B4 receptor 1 (BLT1), to attract and activate leukocytes during inflammation. A role for the LTB4-BLT1 pathway in allergen-induced airway hyperresponsiveness and inflammation is not well defined. OBJECTIVES: To define the role of the LTB4 receptor (BLT1) in the development of airway inflammation and altered airway function. METHODS: BLT1-deficient (BLT1 -/-) mice and wild-type mice were sensitized to ovalbumin by intraperitoneal injection and then challenged with ovalbumin via the airways. Airway responsiveness to inhaled methacholine, bronchoalveolar lavage fluid cell composition and cytokine levels, and lung inflammation and goblet cell hyperplasia were assessed. RESULTS: Compared with wild-type mice, BLT1 -/- mice developed significantly lower airway responsiveness to inhaled methacholine, lower goblet cell hyperplasia in the airways, and decreased interleukin (IL)-13 production both in vivo, in the bronchoalveolar lavage fluid, and in vitro, after antigen stimulation of lung cells in culture. Intracellular cytokine staining of lung cells revealed that bronchoalveolar lavage IL-13 levels and numbers of IL-13(+)/CD4+ and IL-13(+)/CD8+ T cells were also reduced in BLT1 -/- mice. Reconstitution of sensitized and challenged BLT1 -/- mice with allergen-sensitized BLT1 +/+ T cells fully restored the development of airway hyperresponsiveness. In contrast, transfer of naive T cells failed to do so. CONCLUSION: These data suggest that BLT1 expression on primed T cells is required for the full development of airway hyperresponsiveness, which appears to be associated with IL-13 production in these cells.     

Bronchoalveolar lavage cells from sarcoidosis patients and healthy controls can efficiently present antigens.

OBJECTIVES: The interaction between antigen-presenting cells (APC) and T lymphocytes, that recognize the antigen-HLA complex using its T cell-receptor for antigen, is of crucial importance for a subsequent specific immune response. In patients with pulmonary sarcoidosis, the local antigen-presenting capacity in the lungs has been suggested to be abnormally enhanced, and implicated in the immunopathogenesis of the disease. This study was aimed at increasing the understanding of the capacity to present antigens by APC in the lung compartment. DESIGN AND SUBJECTS: We used bronchoalveolar lavage (BAL) cells and paired peripheral blood mononuclear cells (PBMC) of six sarcoidosis patients and two healthy controls to stimulate in total eight well characterized T-cell clones with known HLA and antigen specificities. All subjects were HLA typed. RESULTS: BAL cells of sarcoidosis patients as well as of healthy controls efficiently induced proliferation of the relevant T-cell clone in an HLA-restricted manner when adding either intact antigen or antigenic peptides. CONCLUSIONS: BAL cells have the capacity to process and present antigens adequately, irrespective of whether they are derived from healthy individuals or from patients with sarcoidosis, implying the alveolar space as an important location for active immune reactions.     

Detection of histoplasmal antigens in mice undergoing experimental pulmonary histoplasmosis

A micro-ELISA assay was developed for the quantitation of Histoplasma capsulatum antigen in lungs, bronchoalveolar lavage fluid (BALF), and serum of intranasally infected mice. As little as 0.2 ng of antigen/ml could be detected. During the course of experimental histoplasmosis, immunologically intact, thymus-containing mice (nu/+) had detectable histoplasmal antigens in their lungs, serum, and BALF within 1 day of challenge. Lung, BALF, and serum antigen concentration rose to a peak 2 wk after challenge; in nu/+ mice, antigen concentration then declined through the next 2 wk. In contrast, athymic nude mice have depressed cell-mediated immunity; their antigen concentration continued to rise throughout the course of progressive, ultimately lethal, illness. Antigen concentrations correlated with quantitative cultures of the lungs and BALF. There was little cross reactivity in mice challenged intranasally with Candida albicans or Blastomyces dermatitidis. The sensitivity of this test, and the apparently minimal cross reactivity, suggest that the micro-ELISA for histoplasmal antigen might have significant clinical application in diagnosing and monitoring the course of histoplasmosis.     

Quantitation of cellular and extracellular constituents of the pulmonary lining in rats by using bronchoalveolar lavage. Effects of silica-induced pulmonary inflammation

The efficacy of bronchoalveolar lavage in the removal of cellular and extracellular components of the lining layer from the lungs of silica-treated and control rats was determined. Exponential functions were fitted to curves generated by plotting the quantity of lining layer constituent removed from the lungs by bronchoalveolar lavage versus the lavage number. From these exponential functions we determined the total amount of constituent available in the pulmonary extracellular lining and hence the efficacy of the lavage procedure in removing materials from the lungs. With control rats the removal of extracellular phospholipids, soluble protein, alkaline phosphatase, and beta-N-acetylglucosaminidase by bronchoalveolar lavage occurred at significantly different rates. Removal of 95% of the total available extracellular phospholipid, beta-N-acetylglucosaminidase, soluble protein, and alkaline phosphatase from the lungs required 4, 4, 8, and 11 lavages, respectively. Removal of 95% of the total available alveolar macrophages required 18 lavages. The influence of pulmonary inflammation on the efficacy of the lavage procedure was investigated by injecting silica dust intratracheally into the lungs of rats (50 mg/200- to 250-g rat) and after 3 days performing the analyses. Silica caused an inflammatory condition in the lungs resulting in the accumulation of materials in the alveoli. Highly significant increases in soluble protein (16-fold), alkaline phosphatase (9-fold), and beta-N-acetylglucosaminidase (11-fold), polymorphonuclear leukocytes, eosinophils, and lymphocytes were observed. Alveolar macrophages and extracellular phospholipid were not significantly elevated at 3 days after dosing. Silica did not alter the efficacy of the lavage procedure in removing from the lungs any of the extracellular constituents of the lung lining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of CD4+CD8+ double positive T cells and increase in CD5+ B cells in efferent lymph in sheep infected with Trypanosoma evansi

The effects of Trypanosoma evansi on efferent lymphocyte phenotypes draining from a lymph node primed with Pasteurella haemolytica vaccine were studied in sheep. The prefemoral efferent lymphatic ducts of the infected sheep along with those of two uninfected sheep were surgically cannulated. Lymph was collected and lymphocytes recovered from it analysed by two-colour indirect immunofluorescence staining and cytofluoremetry in a fluorescence activated cell analyser (FACSCAN). The study showed the appearance and persistence of T. evansi in the efferent lymph for a long period of time and the appearance of CD4⁺CD8⁺ (double positive, DP) T lymphocytes in the efferent lymph of infected animals. The infection also resulted in increases in CD5⁺ B cells in the prefemoral efferent lymph. In addition, there were decreases in the output of conventional B cells, CD5⁺ and CD4⁺ T cell subsets but large increases in CD8⁺ cells followed by terminal depletion of all cell subsets. In contrast, inoculation of sheep with pasteurella vaccine antigen alone produced little alterations in the proportions, but large increases in the numbers of all T cell subsets except that of CD8⁺ cells which also showed little variation; and there was a concurrent increase in the numbers and proportions of efferent B cells. In addition, the abnormal expression of DP and CD5⁺ B cells did not occur in the uninfected vaccinated sheep. It is concluded that these abnormal changes in the kinetics of efferent lymphocyte phenotypes are likely to play a role in the genesis of the generalized immunosuppression seen in trypanosome-infected hosts.