Pulmonary immune responses to Nippostrongylus brasiliensis: isotype-specific antibodies in bronchoalveolar lavage fluids of rats.

Larvae of Nippostrongylus brasiliensis have an obligatory migratory phase through the lungs of rats during their development. Since earlier studies have shown that this migration is associated with accumulation of Fc receptor bearing effector cells in the bronchoalveolar spaces, we have analysed antibody reactivity in bronchoalveolar lavage fluids (BALF) during development of immune responses against N. brasiliensis. The development of parasite specific antibodies in bronchoalveolar spaces was similar to that in the serum, but was of a lower titre. A secondary infection resulted in an anamnestic response. Isotype analysis showed that IgG, IgA and IgM antibodies were present in BALF and they recognized several proteins of the parasite ranging from 16-290 kDa. Immunoblot analysis on two-dimensional electrophoretic separated parasitic proteins identified stage specific differences in the BALF antibody responses. IgG was the predominant class of antibody in BALF and when compared with serum, IgM antibody responses were weak. Thus, infection with N. brasiliensis resulted in the appearance of site-, stage- and isotype-specific antibody responses in the lungs of rats.

Pulmonary inflammation and immune responses during the course of Nippostrongylus brasiliensis infection: lymphocyte subsets in bronchoalveolar lavage fluids of rats.

Quantitative measurements were made of different phenotypes of lymphocytes in bronchoalveolar lavage fluid (BALF) of rats during the course of a primary or secondary infection with Nippostrongylus brasiliensis. These changes were compared with those in the peripheral blood to understand the site-specificity of the responses. Following infection, there was a significant increase in both B and T lymphocytes in BALF. The CD4:CD8 ratio was significantly altered with a decreased ratio on day 2 and increased ratio on days 16 and 32 post infection (p.i.). Two colour analysis showed that during larval migration through the lungs (day 2 p.i.) there was a significant increase in CD8+, CD4+ OX22+ and CD4+ OX22- cells in BALF. As infection progressed in time, CD4+ OX22- cells were increased significantly. Compared to primary infection, a secondary infection resulted in increased recovery of CD4+ OX22- cells in BALF. These changes were not readily appreciated in the peripheral blood, suggesting site-specific compartmentalization of lymphocyte responses in the lung. The functional significance of these dynamic changes in lymphocyte subsets in the airspaces following infection remains to be identified.

IgE antibody responses in bronchoalveolar spaces of rats infected with Nippostrongylus brasiliensis.

IgE levels in the bronchoalveolar lavage fluids (BALF) of rats increased significantly following infection with Nippostrongylus brasiliensis. This increase corresponded with a concurrent increase in serum IgE levels. However, a comparison of IgE to albumin ratio in both BALF and serum suggested local accumulation and/or production of IgE in the bronchoalveolar spaces rather than leakage from serum. Subsequent analysis of BALF showed presence of heat-labile PCA activity with highest anti-worm titer (1:64) on Days 11-16 postinfection (pi). Secondary infection resulted in up to a fourfold increase in PCA activity compared to primary infection. Immunoblot analysis showed that these parasite-specific IgE antibodies in BALF recognized many proteins of adult worms ranging from 16-290 kDa. IgE antibodies in serum and BALF showed similarities in their reactivities toward adult worm antigens. However, the IgE antibody reactivities to different antigens varied significantly among different days pi. Depletion of IgG from BALF and serum resulted in more intense binding by IgE antibodies to antigens than when IgG was not depleted. Concurrent with the elevated levels of IgE antibodies, there was a significant increase in the levels of histamine in BALF, suggesting activation of mast cells. Thus, following N. brasiliensis infection there is an abundance of parasite-specific IgE antibodies in the lower respiratory tract and IgE-mediated pathways of inflammation appeared to be activated in the lungs.

Pathology of pulmonary parasitic migration: morphological and bronchoalveolar cellular responses following Nippostrongylus brasiliensis infection in rats.

Nippostrongylus brasiliensis has an obligatory migratory phase through the lungs during its development in rats. This migration is associated with marked tissue damage and pronounced cellular reaction. Given that cells from the lower respiratory tract, especially alveolar macrophages, can adhere to and kill larvae of N. brasiliensis in vitro, we studied the time course of morphological changes associated with parasitic migration. Compared to a primary infection, a secondary infection resulted in significant changes in the pulmonary tissue characterized by an early acute inflammation leading to granulomatous reaction in the parenchyma and a leucocytosis in the bronchoalveolar lavage fluids with an anamnestic increase in absolute numbers of neutrophils, alveolar macrophages, eosinophils, and lymphocytes. Scanning electron microscopy showed that inflammatory cells, especially alveolar macrophages, granulocytes, lymphocytes, erythrocytes, and platelets, adhered to the larvae following secondary infection and this adhesion was associated with disruption of cuticular surface in some larvae. Secondary infection also resulted in retention of larvae in granulomatous lesions in the lungs even up to 21 days postinfection. There was mast cell and type II pneumocyte hyperplasia and these cells appeared to be activated. Thus, the histopathological changes in lungs correlated with the bronchoalveolar cellular responses and further document the inflammatory and immunological reactions during the migration of N. brasiliensis larvae.

Pulmonary inflammation in parasitic infection: immunoglobulins in bronchoalveolar washings of rats infected with Nippostrongylus brasiliensis.

Despite marked pulmonary pathology caused by larval stages of many helminth parasites, little is known about the mechanisms of immune and inflammatory responses to parasites in the respiratory tract. Using bronchoalveolar lavage (BAL) we have retrieved soluble proteins and cells from the respiratory tract of rats given a primary or secondary infection with the nematode Nippostrongylus brasiliensis. Total amounts of different immunoglobulin classes and albumin in BAL fluids and serum were quantitated using an ELISA. Analysis of the cellular component showed an increase in alveolar macrophages, neutrophils, eosinophils and lymphocytes on different days post-infection similar to our earlier findings. A time course study revealed that the concentrations of total protein, albumin, IgG, IgA and IgM in BAL fluids of infected animals were increased from days 2 to 32 after a primary infection. The magnitude of this increase was higher following a challenge infection (secondary) with the same parasite. Moreover, there was also a biphasic increase in total protein, IgG and IgA after secondary infections, with peaks on days 2 to 4 and 11 to 21 post-infection. A comparison of immunoglobulin to albumin ratios in serum and BAL fluids showed that the initial peak of proteins in the lavage was a result of serum leakage and the subsequent peak was due to local secretion of immunoglobulins. These results suggest that in addition to marked BAL cellular reactivity, N. brasiliensis infection induces an initial vascular and endothelial permeability in the respiratory tract which is soon repaired but followed by local synthesis and secretion of IgG and IgA in the lower respiratory tract.

Broncho-alveolar leucocyte responses during primary and secondary Nippostrongylus brasiliensis infection in the rat.

Using broncho-alveolar lavage, we have studied the cellular responses in the rat lung following primary and secondary infection with Nippostrongylus brasiliensis. During the primary infection, there was a biphasic increase in total broncho-alveolar leucocytes and in the absolute numbers of macrophages, neutrophils, eosinophils and lymphocytes. The first peak occurred on days 4-6, and the second peak occurred around day 16, after infection. During the secondary infection there was an anamnestic-like response by all cell types. These data suggest that the broncho-alveolar leucocyte responses to infection have an immunological basis and that in addition to the alveolar macrophage, neutrophils, eosinophils and lymphocytes may play a significant role in lung resistance against migrating helminth larvae.

Complement-dependent killing of Nippostrongylus brasiliensis infective larvae by rat alveolar macrophages.

Histopathological studies have provided circumstantial evidence that helminth parasite destruction occurs in the lung; however controlled in vitro studies on the helminthocidal activity of lung cells have not been reported. This study presents evidence that Nippostrongylus brasiliensis infection in the rat induces alterations in broncho-alveolar lavage (BAL) cell numbers, differential counts, and in vitro helminthocidal activity. Normal, uninfected rats yielded 3.3 +/- 0.6 X 10(6) BAL cells/rat, consisting predominantly of alveolar macrophages (greater than 90%). However on days 2-8 post-infection there was a 1.5-2.4-fold increase in BAL cell numbers with a significant neutrophilia on day 2 and a significant increase in the absolute number of all cell types on day 8. On day 32 post-infection, BAL cell numbers had returned to control levels. Normal BAL cells neither adhered to nor killed N. brasiliensis infective larvae (L3) in the presence of rat complement. By contrast BAL cells recovered from infected rats on days, 2, 8 or 32 post-infection (D2, D8 and D32 BAL cells, respectively) adhered under similar conditions. However, only D8 and D32 BAL cells killed L3. This complement-dependent killing correlated with significantly increased numbers of C3 receptor bearing alveolar macrophages in D8 and D32 BAL cells. Complement-dependent alveolar macrophage helminthocidal activity may therefore play an important role in lung resistance against resident or migrating helminths.