CCSP modulates airway dysfunction and host responses in an Ova-challenged mouse model.

Clara cell secretory protein (CCSP) is synthesized by nonciliated bronchiolar cells in the lung and modulates lung inflammation to infection. To determine the role of CCSP in the host response to allergic airway disease, CCSP-deficient [(-/-)] mice were immunized twice with ovalbumin (Ova) and challenged by Ova (2 or 5 mg/m(3)) aerosol. After 2, 3, and 5 days of Ova aerosol challenge (6 h/day), airway reactivity was increased in CCSP(-/-) mice compared with wild-type [CCSP(+/+)] mice. Neutrophils were markedly increased in the bronchoalveolar lavage fluid of CCSP(-/-) Ova mice, coinciding with increased myeloperoxidase activity and macrophage inflammatory protein-2 levels. Lung histopathology and inflammation were increased in CCSP(-/-) compared with wild-type mice after Ova challenge. Mucus production, as assessed by histological staining, was increased in the airway epithelium of CCSP(-/-) Ova mice compared with that in CCSP(+/+) Ova mice. These data suggest a role for CCSP in airway reactivity and the host response to allergic airway inflammation and provide further evidence for the role of the airway epithelium in regulating airway responses in allergic disease.

Pulmonary immunity to ragweed in a Beagle dog model of allergic asthma.

To create an allergy model in the dog, allergic Beagles with high levels of serum immunoglobulin E (IgE) and eosinophilia were bred; resulting puppies were sensitized to ragweed by intraperitoneal (IP) injection within 24 hours of birth through 22 weeks of age. At least 50% of the puppies developed high levels of serum IgE and eosinophilia. As young adults, 6 of these dogs, and 6 control age-matched, nonallergic, nonimmunized dogs were exposed by inhalation to ragweed twice at 13-day intervals, and a third time 45 days later. Total and ragweed-specific serum IgE and ragweed-specific serum IgG were increased significantly in allergic dogs relative to baseline. Allergic dogs had significantly greater levels of antibody specific for ragweed, as well as higher eosinophil counts in the bronchoalveolar lavage fluid, compared to nonallergic dogs. Airway reactivity to histamine in allergic, but not nonallergic dogs, increased significantly after aerosol exposure to ragweed. After a third exposure to ragweed, airway responses to histamine were elevated in the allergic dogs and remained high for at least 5 months. These results demonstrate the potential of the allergic dog model for investigating the underlying pulmonary immune mechanisms and therapeutic treatment of allergic asthma.

Ovalbumin aerosols induce airway hyperreactivity in naïve DO11.10 T cell receptor transgenic mice without pulmonary eosinophilia or OVA-specific antibody.

The pathobiology of allergic asthma is being studied using murine models, most of which use systemic priming followed by pulmonary challenges with the immunizing antigen. In general, mice develop eosinophilic pulmonary inflammation, increased antigen-specific immunoglobulins, and airway hyperreactivity (AHR), all of which are dependent on antigen-specific T cell activation. To establish a model of allergic asthma, which did not require systemic priming, we exposed DO11.10 T cell receptor transgenic mice, which have an expanded repertoire of ovalbumin (OVA), peptide-specific T cells, to limited aerosols of OVA protein. DO11.10 +/- mice developed AHR in the absence of increases in total serum IgE, OVA-specific IgG, or eosinophilia. The AHR was accompanied by pulmonary recruitment of antigen-specific T cells with decreased expression of CD62L and CD45RB and increased expression of CD69, a phenotype indicative of T cell activation. Our results support recent hypotheses that T cells mediate AHR directly.

Animal models of asthma: potential usefulness for studying health effects of inhaled particles.

Asthma is now recognized to be a chronic inflammatory disease that affects the whole lung. Incidence appears to be increasing despite improved treatment regimens. There is substantial epidemiological evidence suggesting a relationship between the incidence and severity of asthma (e.g., hospitalizations) and exposure to increased levels of air pollution, especially fine and ultrafine particulate material, in susceptible individuals. There have been a few studies in animal models that support this concept, but additional animal studies to test this hypothesis are needed. However, such studies must be performed with awareness of the strengths and weaknesses of the currently available animal models. For studies in mice, the most commonly used animal, a broad spectrum of molecular and immunological tools is available, particularly to study the balance between Th1 and Th2 responses, and inbred strains may be useful for genetic dissection of susceptibility to the disease. However, the mouse is a poor model for bronchoconstriction or localized immune responses that characterize the human disease. In contrast, allergic lung diseases in dogs and cats may more accurately model the human condition, but fewer tools are available for characterization of the mechanisms. Finally, economic issues as well as reagent availability limit the utility of horses, sheep, and primates.

Nedocromil sodium inhibits canine adenovirus bronchiolitis in beagle puppies.

Nedocromil sodium is a nonsteroidal anti-inflammatory drug used to control asthmatic attacks. Our hypothesis is that nedocromil sodium inhibits virus-induced airway inflammation, a common trigger of asthma. We nebulized nedocromil sodium into beagle dogs (n = 10, mean +/- SEM ages: 149 +/- 13 days) before and after inoculation with canine adenovirus type 2 (CAV2). Control dogs (n = 10) received saline aerosols and were either infected with CAV2 (Sal/CAV2, n = 7, mean +/- SEM ages: 140 +/- 11 days) or were not infected (Sal/Sal, n = 3, ages: 143 +/- 0 days). All dogs were anesthetized with choralose (80 mg/kg i.v.), intubated, and mechanically ventilated. Pulmonary function tests and bronchoalveolar lavage (BAL) were performed using standard techniques. Pulmonary function tests revealed no significant change between the nedocromil sodium and non-nedocromil-treated groups. The percentage of infected bronchioles was quantitated as the number of inflamed airways of 40 bronchioles examined times 100 for each dog. Nedocromil-treated dogs had significantly (p < 0.05) less mucosal inflammation (mean +/- SEM, 39% +/- 5%), epithelial denudation (36% +/- 5%), and BAL neutrophilia (11 +/- 3) than did Sal/CAV2 dogs (51% +/- 6%, 57% +/- 4%, and 33% +/- 8%, respectively). We concluded that pretreatment with nedocromil sodium aerosols attenuated CAV2-induced airway inflammation in these beagle puppies.

Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells.

A hallmark of asthma is mucin overproduction, a condition that contributes to airway obstruction. The events responsible for mucin overproduction are not known but are thought to be associated with mediators of chronic inflammation. Others have shown that T-helper 2 (Th2) lymphocytes are required for mucous cell metaplasia, which then leads to mucin overproduction in animal models of allergy. We hypothesized that Th2 cell mediators are present in asthmatic airway fluid and directly stimulate mucin synthesis in airway epithelial cells. Results in cultured airway epithelial cells showed that samples of asthmatic fluid stimulated mucin (MUC5AC) synthesis severalfold more potently than non-asthmatic fluid. Consistent with this, lavage fluid from the airways of allergen-challenged dogs stimulated mucin synthesis severalfold more potently than that from non-allergen-challenged dogs. Fractionation of dog samples revealed 2 active fractions at <10 kDa and 30-100 kDa. Th2 cytokines in these molecular weight ranges are IL-9 (36 kDa), IL-5 (56 kDa), and IL-13 (10 kDa). Antibody blockade of ligand-receptor interaction for IL-9 (but not IL-5 or IL-13) inhibited mucin stimulation by dog airway fluid. Furthermore, recombinant IL-9, but not IL-5 or IL-13, stimulated mucin synthesis. These results indicate that IL-9 may account for as much as 50-60% of the mucin-stimulating activity of lung fluids in allergic airway disease.

Dissociation of airway hyperresponsiveness from immunoglobulin E and airway eosinophilia in a murine model of allergic asthma.

Nonspecific airway hyperresponsiveness (AHR) is a hallmark of human asthma. Both airway eosinophilia and high serum levels of total and antigen-specific immunoglobulin E (IgE) are associated with AHR. It is unclear, however, whether either eosinophilia or increased IgE levels contribute directly to, or predict, the development of AHR. Investigations conducted with various murine models of asthma and different mouse strains have resulted in conflicting evidence about the roles that IgE and airway eosinophilia play in the manifestation of AHR. We show that systemic priming with ovalbumin (OVA) in alum, followed by a single day of OVA aerosol challenge, is sufficient to induce AHR, as measured by increased pulmonary resistance in response to intravenously delivered methacholine in BALB/c, but not C57BL/6 or B6D2F1, mice. This was observed despite the fact that OVA-challenged BALB/c mice had less airway eosinophilia and smaller increases in total IgE than either C57BL/6 or B6D2F1 mice, and had less pulmonary inflammation and OVA-specific IgE than B6D2F1 mice. We conclude that airway eosinophilia, pulmonary inflammation, and high serum levels of total or OVA-specific IgE are all insufficient to induce AHR in C57BL/6 and B6D2F1 mice, whereas BALB/c mice demonstrate AHR in the absence of airway eosinophilia. These data confirm that the development of AHR is genetically determined, not only in naive mice, but also in actively immunized ones, and cannot be predicted by levels of airway eosinophilia, pulmonary inflammation, total IgE, or antigen-specific IgE.

Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs.

In ragweed (RW)-sensitized beagle dogs, we tested the hypothesis that reactivity of the pulmonary vasculature was enhanced with aerosolized histamine (Hist) and RW. Seven dogs were neonatally sensitized with repeated intraperitoneal RW injections, and 12 dogs were controls (Con). The dogs were anesthetized with intravenous chloralose, mechanically ventilated, and instrumented with femoral arterial and pulmonary artery catheters. Specific lung compliance (CLsp), specific lung conductance (Gsp), systemic vascular resistance index, and pulmonary vascular resistance index (PVRI) were measured before and after bronchoprovocation with Hist and RW. After Hist inhalation (5 breaths of 30 mg/ml), both Con and RW dogs had significant (P < 0.05) decreases in CLsp (-51 +/- 4 and -53 +/- 5%, respectively) and Gsp (-65 +/- 5 and -69 +/- 3%, respectively), but only RW-sensitized dogs had a significant increase in PVRI (38 +/- 10%). After RW inhalation (60 breaths of 0.8 mg/ml), only RW-sensitized dogs had significant increases (62 +/- 20%) in PVRI and decreases in Gsp (-77 +/- 4%) and CLsp (-65 +/- 7%). We conclude that, compared with Con, RW-sensitized beagle dogs have increased pulmonary vasoconstrictive responses with Hist or RW inhalation.

Immunoglobulin response to intrapulmonary immunization of asthmatics.

Atopic asthmatics, compared to non-atopic individuals, exhibit an increased amount of serum antigen-specific IgE and IgG4 antibody directed toward many aeroallergens. We tested the hypothesis that this difference between atopics and non-atopics extends to the response to intrapulmonary deposition of a neoantigen, keyhole limpets haemocyanin (KLH). We immunized nine atopic asthmatics and nine non-atopic controls with 500 micrograms KLH instilled into a subsegment of the lingula and examined serum anti-KLH, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM and specific antibody production by peripheral blood mononuclear cells for 25 days. We also determined specific antibody in bronchoalveolar lavage fluid (BALF) in both the immunized and a non-immunized lobe 11 days after immunization. We found specific serum antibody in all immunized subjects with no difference between atopics and normals in the amount or kinetics of anti-KLH IgG1, IgG2, IgG3, IgA1, IgA2 and IgM. However, the atopics exhibited more anti-KLH IgG4 than the normal controls. Specific anti-KLH antibody-producing cells were detected in peripheral blood in most subjects at day 8 to 12 after immunization with no difference between atopics and normals. Specific IgA1, IgA2, IgG1 and IgM antibodies were detected in BALF from the immunized lobes but not from the non-immunized lobes of both groups of subjects with no difference between atopics and normals. We conclude that atopic asthmatics respond to intrapulmonary KLH with more serum anti-KLH IgG4 than normal controls, consistent with a bias toward a Th2 response to intrapulmonary exposure to antigen.

Neutrophil involvement in the retention and clearance of dust intratracheally instilled into the lungs of F344/N rats.

This study evaluated polymorphonuclear leukocyte (PMN) involvement in translocation of dust to bronchial lymph nodes after deposition of dust in the lungs of control and neutropenic F344/N rats. Rats were rendered neutropenic with an intraperitoneal (IP) injection of anti-rat PMN antiserum (APA); control rats were injected IP with 0.9% saline solution. Eighteen hours after IP injections, control and APA-treated rats were instilled intratracheally with 5 x 10(8) microspheres suspended in 0.9% saline solution, which caused an influx of PMNs into the pulmonary airspaces of control rats, but not of APA-treated rats. One day postinstillation (PI), 77.2% of the microspheres recovered in bronchoalveolar lavage fluid (BALF) from control rats were associated with pulmonary alveolar macrophages (PAMs) and 18.8% with PMNs; 4.0% were free. In BALF from the APA-treated rats, 66.3% of the microspheres were associated with PAMs and 0.3% with PMNs; 36.3% were free. Two days PI, about 95% of the microspheres in BALF from control and APA-treated rats were associated with PAMs; by 4 and 7 days PI, essentially 100% were with PAMs. Amounts of microspheres translocated to bronchial lymph nodes of control rats were four fold less than in the APA-treated rats on days 2, 4, and 7 PI (p < .05). The results suggest that PMNs in pulmonary airspaces of F344/N rats phagocytize dust and thereby interfere with the mechanism(s) involved in dust penetration into the pulmonary interstitium.

Evaluation of association of blood and bronchoalveolar eosinophil numbers and serum total immunoglobulin E concentration with the expression of nonspecific airway reactivity in dogs.

OBJECTIVE: To characterize the relation between bronchoalveolar and blood eosinophil numbers, serum total IgE concentration, and nonspecific airway reactivity in healthy dogs. ANIMALS: 26 healthy Beagles. PROCEDURE: Prior to measurement of nonspecific airway responsiveness, dogs were anesthetized and bronchoscopy was performed to recover bronchoalveolar lavage (BAL) fluid. Repeated measurements were made in 6 dogs. RESULTS: The percentage of blood eosinophils varied between 0 and 13 (mean +/- SD, 5.6 +/- 3.6) %, the percentage of eosinophils in BAL fluid ranged between 0 and 63.5 (8.8 +/- 12.9) %, and total serum IgE concentration was 0.1 to 107.5 (23.4 +/- 29.1) U/ml. A strong association was evident between numbers of blood eosinophils and total serum IgE concentration (R2 = 0.413, P < 0.001), and a trend toward an association between numbers of blood eosinophils and numbers of eosinophils in BAL fluid was apparent (R2 = 0.110, P < 0.053). Significant associations were not found between any other aspects of the blood and BAL fluid cell profiles and total serum IgE concentration or airway reactivity. Serum total IgE concentration was not associated with airway reactivity. Further, in dogs examined on repeated occasions, variation in BAL fluid eosinophil numbers was not associated with any change in serum total IgE concentration or airway reactivity. CONCLUSIONS: Neither numbers of bronchoalveolar or blood eosinophils nor serum total IgE concentration have a significant role in determining airway reactivity in health dogs.

Pulmonary immune memory: localized production of antibody in the lung after antigen challenge.

In comparison to primary immune responses after lung immunization, the level of antigen-specific antibody and the number of cells producing specific antibody are significantly increased after challenging the lungs with antigen. The response of immune memory cells in the lung to an antigen challenge could be responsible for this elevated immune response. However, increased numbers of antibody-producing cells, possibly produced in the lung-associated lymph nodes, are also found in the blood after an antigen challenge. Therefore, it is possible that both the response of immune memory cells in the lung, and the recruitment of antibody-producing cells from the blood, contribute to the elevated levels of antibody in the lung after an antigen challenge. This study compared the level of antibody produced in the lung by the response of pulmonary immune memory cells with the level of antibody produced by antibody-forming cells that enter the lung from blood after an antigen challenge. This comparison was made possible by immunizing and challenging two lung lobes of dogs with two antigens. The immune responses to both antigens were then evaluated in both lung lobes after primary immunization and challenge. Data from these evaluations showed that most antibody in the lung after an antigen challenge is produced by a localized anamnestic response of pulmonary immune memory cells. A significantly lower level of antibody entered the lung from the vasculature and/or was produced by antibody-forming cells that entered the lung from blood after an antigen challenge.

Long-term antibody production in canine lung allografts: implications in pulmonary immunity and asthma.

Lung transplant recipients can become asthmatic if they receive donor lungs from asthmatics. The maintenance of sensitivity in the lung allograft for inhaled allergens supports the concept that the mechanisms responsible for asthma are localized in the lungs, with a minimal systemic component. Pulmonary immunity to inhaled allergens is one mechanism which could be localized to the lung that would play a pivotal role in asthma. For example, the continued production of antibody to inhaled allergens in a human lung allograft could cause asthmatic responsiveness in the recipient. In this study, we tested the hypothesis that pulmonary immune cells continue to produce antibody in a canine allograft lung for relatively long times after transplantation. This was accomplished by immunizing four dogs by instillation of keyhole limpet hemocyanin (KLH) into a single lung lobe. After two challenges, the immunized lung from each dog was transplanted into a nonimmune recipient. Immune evaluations of recipients showed that anti-KLH antibody continued to be produced only in the donor lung for as long as 320 days after transplantation. Data from this study suggest that (1) immune cells in the lung can function independently from systemic immunity, (2) antibody production in the lung makes a significant contribution to blood antibody levels, and (3) immune cells in donor lungs can continue to produce antibody for relatively long times after transplantation. Therefore, immune cells in donor lungs from asthmatics could continue to produce antibody to allergens after transplantation, and this locally produced antibody may be responsible for the asthmatic responses observed in the recipients.

The regulation of pulmonary immunity.

No evidence has emerged which suggests that the principles of immunity derived from studies on cells from other body sites are contradicted in the lung and its associated lymphoid tissue. What is clear, however, is that the environment dictates the types of cells, their relationship to one another, and what perturbing events will set in motion either the development of an "active" immune response or tolerance. Investigating mechanisms for the development of lung immunity has increased our understanding of how human diseases develop and is continuing to suggest new ways to manipulate pulmonary immune responses. Demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immunosuppressed hosts, and to suppress manifestations of immunologically mediated lung disease. Important lung diseases targeted for intensive research efforts in the immediate future are tuberculosis, asthma, and fibrotic lung disease. Perhaps even the common cold might be conquered. Considering the pace of current research on lung immunity, it may not be too ambitious to predict that these diseases may be conquered in the next decade.

Intrapulmonary antigen deposition in the human lung: local responses.

We hypothesized that, as in animal models, localized deposition of antigen into the human lung would induce local inflammatory and immune responses in antigen-exposed sites. To test this hypothesis, segmental instillation of a well-characterized, highly immunogenic, soluble antigen, keyhole limpet hemocyanin (KLH) was performed in 10 healthy, nonsmoking volunteers. Ten to fifteen days after instillation, bronchoalveolar lavage (BAL) was performed in immunized segments (IS) and contralateral control segments (CS) and local responses to antigen instillation were assessed by comparing IS and CS BAL. Greater albumin concentrations and cell recoveries were found in IS than in CS BAL, suggesting local inflammation. Although total numbers of each cell type were increased, relative proportions of alveolar macrophages, lymphocytes, and neutrophils were similar in IS and CS BAL. CD4/CD8 ratios in IS BAL samples were greater than those in CS samples, because of higher numbers of CD4+ lymphocytes in IS than in CS BAL but similar numbers of CD8+ lymphocytes. Anti-KLH IgG and IgA concentrations were greater in IS than in CS BAL. However, anti-KLH IgG/albumin ratios were similar in IS BAL and serum, suggesting that anti-KLH IgG had reached IS by passive transudation from the circulation. In contrast, anti-KLH IgA/albumin concentrations were greater in IS BAL than in serum, suggesting local production, and/or active transport of serum-derived anti-KLH IgA into the IS. Fractionation of serum and IS BAL on sucrose gradients demonstrated that anti-KLH IgA activity was largely associated with 11S polymeric IgA in both locations.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat-killed pneumococci and pneumococcal capsular polysaccharides stimulate tumor necrosis factor-alpha production by murine macrophages.

Tumor necrosis factor-alpha (TNF) is an important humoral mediator of sepsis and endotoxin-induced shock. However, Streptococcus pneumoniae, a gram-positive organism, is the most common causative agent of community-acquired pneumonia and sepsis. We hypothesized that the pathogenesis of pneumococcal pneumonia and sepsis involves pneumococcus-stimulated TNF synthesis, and we tested that hypothesis in vitro by comparing heat-killed type III and type V pneumococcus and 23-valent purified pneumococcal capsular polysaccharides with Escherichia coli and purified lipopolysaccharide (LPS) as stimuli for TNF production by the murine macrophage cell line RAW 264.7. We evaluated TNF production in response to various doses and times of exposure to these agents, as well as the effects of indomethacin on TNF production in response to these agents. Stimulation with both types of heat-killed pneumococcus resulted in TNF production in a dose-response fashion, as did stimulation with E. coli. Fewer type III pneumococci (10 bacteria/ml) were required to stimulate significant TNF secretion than either type V pneumococcus or E. coli, but the overall dose-response curves of the three bacteria were similar. The dose-response curves for pneumococcal capsular polysaccharides and LPS were very similar, although at the highest concentration pneumococcal capsular polysaccharides stimulated more TNF secretion than did LPS (469 versus 213 U/ml). The kinetics of pneumococcus-stimulated TNF secretion were identical to the kinetics of LPS-stimulated TNF secretion. In the presence of indomethacin, pneumococcus-stimulated TNF production decreased by 87.5%, as compared with pneumococcus alone. In contrast, LPS with indomethacin stimulated 19.5% more TNF than LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term antibody production after lung immunization and challenge: role of lung and lymphoid tissues.

After localized lung immunization and challenge, antigen-specific antibody continues to be produced in the immunized lung lobes of dogs for years after the last antigen exposure. Lavage fluid from immunized lung lobes contains significantly more antigen-specific antibody than lavage fluid from control lung lobes, and only cells from lung lobes exposed to antigen produce antibody. Although cells lavaged from the lung produce antibody, it is possible that cells in the lung interstitium or lymphoid tissues may be more important in long-term antibody production after lung immunization and challenge. The goal of this study was to compare the levels of antibody production by cells from dogs 2 yr after pulmonary immunization and challenge. Cells were evaluated from lung lavage, lung tissue, tracheobronchial lymph nodes, and distant lymphoid tissues. The results showed that cells lavaged from lung lobes immunized and challenged with sheep red blood cells (SRBC) were producing anti-SRBC IgG antibody 2 yr after the last antigen challenge. However, cells obtained by mincing tissues from immunized lung lobes were producing significantly higher levels of antibody than lavage cells. In contrast, lavage or tissue cells obtained from the control lobes did not produce detectable antibody. Only a low level of anti-SRBC IgG was produced by cells from the tracheobronchial lymph nodes, and minimal antibody was produced by cells from blood, spleen, or mesenteric and popliteal lymph nodes.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of age on immune responses in the antigen-instilled dog lung. Antibody responses in the lung and lymphoid tissues following primary and secondary antigen instillation.

To evaluate the effects of age on immunity induced by lung immunization, 11 aged (12-17 years; median age = 14) and 12 young (2-5 years) male Beagle dogs were instilled with 10 mg of keyhole limpet hemocyanin (KLH) in the right cardiac lung lobe and 10(10) sheep red blood cells (SRBC) in the left cardiac lung lobe. Five aged and six young dogs were sacrificed at day 9 after primary antigen instillation. The remainder were given challenge antigen instillations of KLH and SRBC at day 21 and sacrificed 7 days later. Serum, bronchoalveolar lavage fluid and lung tissue from immunized and control lobes, tracheobronchial, mesenteric and popliteal lymph nodes, spleen, and blood were taken at sacrifice. Anti-KLH IgA, IgG and IgM antibody production by cells in lung tissue and lavage fluid from the KLH-exposed lobe was lower at primary immunization and challenge in aged than young dogs. Lavage fluid IgA and IgG levels from the KLH exposed lobe at primary immunization and challenge were lower in aged versus young dogs, while IgM levels were lower only after primary immunization. Localized lung immune memory responses were also markedly lower in aged dogs when compared with young dogs. Anti-SRBC responses were similar to the anti-KLH responses. Our data show that systemic immune responses are significantly lower in aged dogs following primary antigen instillation, but not after antigen challenge in the lung. This was not the case for localized lung immune responses, which were significantly lower in aged dogs even following antigen challenge. The data also show that antibody production by lavage cells is a good index of interstitial lung cell antibody production.