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

Mechanisms of Ageing and Development, 68 (1993) 191-207

191

Elsevier ScientificPublishers Ireland Ltd.

THE EFFECTS OF AGE ON IMMUNE RESPONSES IN THE ANTIGENINSTILLED DOG LUNG. ANTIBODY RESPONSES IN THE LUNG AND LYMPHOID TISSUES FOLLOWING PRIMARY AND SECONDARY ANTIGEN INSTILLATION

SUSAN E. JONES*'**, DONNA R. DAVILA *'t, PATRICK J. HALEY ~ and DAVID E. BICE Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, NM 87185 (USA)

(Received May 26th, 1992) (Revision receivedDecember 14th, 1992)

SUMMARY 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 t° 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 imCorrespondence to: David E. Bice,InhalationToxicologyResearchInstitute,P.O. Box 5890, Albuquerque, NM 87185, USA. *Associated WesternUniversitiesPostdoctoralFellowat the time of this study. **Present address: PurdueUniversity,Schoolof VeterinaryMedicine,WestLaFayette,IN 47907, USA. *Present address: Universityof New Mexico,Collegeof Pharmacy,Albuquerque,NM 87137, USA. *Present address: SterlingDrug, Inc., 9 Great ValleyParkway,Malverne,PA 19355, USA.

192

mune 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.

Key words: Immune compartments; Dog; Lung; Antibodies; Age

INTRODUCTION

It is generally accepted that several changes occur in the immune system during the aging process [1-5]. Elderly humans have an increased incidence of pneumonia and other infectious diseases as well as increased incidences of cancer and degenerative diseases [6-11]. These changes are thought to be related to a decline in effective immune responses with advancing age. Previous studies have shown that many aspects of the systemic immune response decline with age [1-5,12]. It is also known that primary antigen-specific antibody responses in bronchoalveolar lavage fluid (BALF) decline with age [12]. No previous study has examined age-related changes in antigen-specific antibody production by lung tissue cells. The dog was chosen for evaluating the effects of age on pulmonary immunity for several reasons. The pulmonary immune responses of the dog are similar to those of humans; hence, the dog serves as an excellent model for studying human pulmonary immune responses [13]. Also, because of the size of the dog, a specific airway can be selected for instillation and repeated lavage without adverse effects. Lastly, patterns of maturation and senescence of the dog are more similar to humans than other commonly used laboratory animals; thus, the dog may be more suitable as a model for human aging. The purpose of this study was to answer the following question: Are there significant age-related differences in systemic, regional and local antibody immune responses following primary and secondary antigen instillation in the lungs? To determine if age is associated with alterations in immune responses to antigen deposited in the lung, the following evaluations were made: (i) systemic and lavage fluid antibody levels, (ii) antibody production by lavage cells and cells from lung tissue, (iii) antibody production by blood lymphocytes and (iv) antibody production by distant and tracheobronchial lymph nodes (TBLN). Two T-cell-dependent antigens were used in this study: KLH, a soluble antigen and SRBC, a particulate antigen. The SRBC antigen was used to produce an immune response in a lung lobe other than the KLH-instilled lobe so that localized immune memory responses could be measured following challenge antigen instillation [14-17].

193

MATERIALS AND METHODS

Care of Beagle dogs Eleven aged (12-17 years old) and 12 young (2-3 years old) male Beagle dogs from the Inhalation Toxicology Research Institute (ITRI) colony were used in this study. The mean age of the aged dogs was 14 years, approximately the median life expectancy of Beagle dogs in the ITRI colony. The dogs were housed in indooroutdoor facilities at ITRI. They were fed once a day with 12 oz of dry kibble dog food (Teklad Premier Laboratory Diets, Madison, WI) and water was available at all times. None of the animals had been exposed to KLH or SRBC previously. Prior to being assigned to the study, all dogs were found to be in good health by physical examination, chest radiographs, serum chemistries and complete blood counts. No dogs showed evidence of respiratory disease.

Preparation of antigen for instillation Keyhole limpets were obtained from Pacific Biomarine, Venice, CA and the KLH used in this study was prepared according to the procedure of Vandenbark [18]. Briefly, each limpet was rinsed with sterile saline and its foot rubbed with a gloved hand. Hemolymph was then collected from the limpets overnight at 4°C in an endotoxin-free aluminum pan. KLH was prepared from crude hemolymph by differential ultracentrifugation [18]. The pelleted KLH was dissolved in normal saline, dialyzed repeatedly at 4°C against normal saline and filtered through a 0.45-/zm filter. Endotoxin levels in the collected hemolymph, tested by the limulus amoebocyte lysate assay (E-toxate, Sigma Chemical Co., St. Louis, MO), were less than 10 ng/ml. The SRBC were collected into a sterile blood collection vial with equal parts Alsevers solution and blood from the jugular vein of a sheep. The SRBC were pelleted using 30-60 ml of the SRBC-Alsevers mixture in a Sorvall refrigerated centrifuge at 200 x g for 15 min. The SRBC were resuspended in cold sterile saline, washed repeatedly and resuspended to a final concentration of 1 × 101°/ml SRBC.

Transbronchoscopic instillation of antigen The dogs were immunized by transbronchoscopic instillation using a fiberoptic bronchoscope (Olympus Corp., model BF, type 4B2, Lake Success, NY) [12]. Food was withheld for 18 h prior to anesthesia. The dogs were anesthetized with halothane and intratracheally intubated; the fiberoptic bronchoscope was then directed into the bronchial airway of the desired lung lobe. A polyethylene catheter (1.57 mm, outer diameter) was passed through the suction port of the bronchoscope into the airway and advanced until resistance was met. All dogs were instilled with 10 mg of KLH in 1 ml of saline (0.9% NaCI solution) in their right cardiac lung lobe (RCL), 1 x 10 l° SRBC in 1 ml of saline in their left cardiac lung lobe (LCL) and 1 ml of saline in their right intermediate lung lobe (RIL). Five aged and six young dogs were

194 then sacrificed 9 days following this primary antigen instillation. The remaining 12 dogs were challenged by instillation of 10 mg of KLH into the RCL, 1 x 101° SRBC into the LCL and saline into the RIL at day 21 following the primary antigen instillation. These dogs were sacrificed at day 7 following the challenge antigen instillation. Day 9 following primary antigen instillation and day 7 following challenge antigen instillation were chosen because previous studies showed that peak antibody responses in BALF corresponded with these days [19-21].

Bronchoalveolar lavage of dogs Bronchoalveolar lavage was performed in all dogs just prior to sacrifice [12]. The dogs were anesthetized with halothane, intubated and a fiberoptic bronchoscope (5.5 mm, outer diameter) was passed into the bronchial airway of the desired lung lobe. The bronchoscope was then wedged in the desired bronchial airway in order to provide good return of lavage fluid. Lavages were performed using five, 10-ml aliquots of normal saline (0.9% NaCI solution) with each aliquot injected through the suction channel of the bronchoscope and then recovered by applying gentle suction with the syringe. Blood was collected by venipuncture (collected for serum in sterile glass tubes without anticoagulant and for cells in tubes containing EDTA) at day 9 for each dog sacrificed after primary antigen instillation and day 7 after the challenge instillation. BALF was collected from the LCL, RCL and RIL lung lobes of each dog on the same days as noted for the blood collection.

Preparation of bronchoalveolar lavage and blood cells BALF from each lung lobe was separated into supernatant and cells by centrifugation at 200 x g for 10 min at 4°C. Supernatant samples were decanted and stored at -20°C. The cell pellets were washed twice in incomplete (no serum added) RPMI1640 medium (GIBCO, Grand Island, NY) and resuspended in complete RPMI (10% fetal bovine serum added). Cells were counted using a Coulter ZBI cell counter (Coulter, Hialeah, FL) and aliquots were removed to make a final concentration of 1 x 10 6 cells/ml in 2.5 ml. Cytocentrifuge slides of the diluted lavage cells were also made for differential cell counts. Blood collected into EDTA tubes was diluted 1:1 with incomplete media, layered over ficoll-hypaque (Isolymph, Gallard-Schlesinger, Carle Place, NY) and centrifuged at 200 x g for 30 min at room temperature. The leukocyte-rich layer was collected and washed three times by centrifugation (200 x g; 10 min; 10°C) in incomplete RPMI and then resuspended to a concentration of 1 x 106 cells/ml. Cytocentrifuge slides of the diluted blood lymphocytes were made for differential cell counts. The remaining blood in the EDTA tubes was used for white blood cell counts and blood smears were made for differential cell counts.

Preparation of tissues for measurement of in vitro antibody production The dogs were injected with pentobarbital to induce deep surgical anesthesia and

195

were then exsanguinated by cardiac puncture. Tissues from the LCL, RCL and RIL, left and right TBLN, which drain the LCL and RCL, respectively [22], spleen, mesenteric lymph node and a popliteal lymph node were taken from each dog. Lung tissue from each lobe was scraped free from the airways and suspended in incomplete RPMI. This fluid and tissue mixture was then sieved through a fine wire mesh screen using a pestle, creating a cellular fluid and minced tissue mixture. Next, this mixture was passed through a 70-micron filter to remove any remaining tissue fragments. Finally, the mixture was layered over a density gradient (ficoll-hypaque), separated and washed as described for peripheral blood. The pelleted cells were resuspended in 10 ml of complete RPMI and aliquots were removed to make a final concentration of 1 x 106 cells/ml in 2.5 ml. Lymph nodes were processed in Dounce tissue grinders (Wheaton, Millville, N J) for preparation of cell suspensions. Cells were washed twice in incomplete RPMI and resuspended in 10 ml of complete RPMI. Cells from these final suspensions were counted, and aliquots were removed to make a final concentration of 1 x 106 cells/ml in 2.5 mi. Spleen portions were cut lengthwise and the cut surface was scraped with a No. 10 scalpel blade. The cellular material was then layered over a density gradient (ficoll-hypaque), separated and washed as described for peripheral blood. Cells in these final suspensions were counted and aliquots were removed to make a final concentration of 1 × 106 cells/ml in 2.5 ml. In vitro immunoglobulin production was assessed by culturing cells, using complete RPMI media, from blood, BALF, LCL, RCL, and RIL tissues, left and right TBLN, spleen and mesenteric and popliteal lymph nodes in round-bottom, 96-well microtiter plates. Parallel cultures were performed with and without 50 t~g/ml cycloheximide. Cultures were incubated at 37°C for 7 days in the microtiter plates. The plates were centrifuged at 200 × g for 15 min at 4°C and cell-free supernatants were removed and stored at -20°C. Cytocentrifuge slides were made from each cellular preparation for each dog. These slides, along with blood smears and bronchoalveolar lavage cell smears, were then stained with Diff-Quick solutions I and II (American Scientific Products, McGaw Park, IL) for differential cell counts. Enzyme-linked immunosorbant assay An enzyme-linked immunosorbant assay (ELISA) was used to determine antiKLH and anti-SRBC antibody responses. KLH antigen used in ELISA evaluations was the same as for immunization. Soluble SRBC antigen prepared from SRBC membranes [23] solubilized with 0.1% sodium dodecyl sulfate was used in ELISA evaluations. Antigens were coated onto 96-well polyvinyl chloride 'U' well microtiter plates (Dynatech, Chantilly, VA) using a phosphate buffered saline solution (Sigma, St. Louis, MO), pH 7.4, as a coating buffer and the plates were placed in a refrigerator at 4°C overnight. The antigens were added to the plates in the minimum concentration (1 #g KLH and 5 #g SRBC per well) that provided reproducible results as determined by preliminary evaluations. The plates were then washed five times

196

with a phosphate buffered saline wash solution, using a 12-channel microtiter well dispenser/aspirator (Nunc-Immuno Wash 12, VWR Scientific, Philadelphia, PA). This wash procedure was repeated before the addition of any other substance to the plates. The sample was added next at the desired dilution using a 2.5% milk (Difco skim milk, CMS, Indianapolis, IN) solution as the diluent. Next, either heavy chainspecific Goat anti-Dog IgG, IgM, (Kirkegaard and Perry, Gaithersburg, MD) or Rabbit anti-Dog IgA (Fc) (Nordic Labs, Capistrano Beach, CA) was added, followed by an enzyme-conjugated anti-immunoglobulin (Kirkegaard and Perry, Gaithersburg, MD) directed against the species in which the antibody was produced. A colorless substrate (ABTS 2,2'-azino-bis [3-ethylbenthiazoline 6-sulfonic acid], Kirkegaard and Perry, Gaithersburg, MD) was added to quantitate bound enzymelabeled anti-immunoglobulin. Optical density (O.D.) units were determined with a microtiter plate reader (Dynatech MR700 microplate reader, Chantilly, VA) using a 410-nm filter, and antibody concentrations were reported as O.D. units. To provide comparative data, ELISA units were determined using the same dilution for all samples in each group (e.g. serum, BALF, or culture supernatants). The dilutions selected gave ELISA O.D. measurements of less than 2.0. Because lymph node size and spleen and lung tissue section sizes varied from dog to dog, tissue ELISA O.D. units were standardized by the following formula: ELISA O.D. = standard ELISA O.D. (cells/well) (% lymphocytes) The Student's t-test was used to estimate all P-values shown unless otherwise noted. Only P-values of _<0.05 were considered significant. Total IgG and IgM antibody in serum was measured for both aged and young dogs. A double antibody sandwich ELISA was used and O.D. readings were converted to known units (mg/ml) based on a standard curve. RESULTS Serum responses Aged dogs had significantly lower serum IgA and IgM anti-KLH antibody levels than young dogs after primary antigen instillation (Fig. I). There were no significant differences between aged and young dogs in serum IgG anti-KLH antibody levels. No significant differences between aged and young dogs were found in serum antiKLH responses following challenge antigen instillation (Fig. 1). Lavage responses Lavage fluid IgA, IgG and IgM anti-KLH antibody responses in the RCL were significantly lower in aged than in young dogs following primary antigen instillation

197

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Fig. 1. Serum anti-KLH IgM, IgG, IgA antibody responses (~ 4- S.E.M.) in aged and young dogs following primary and challenge antigen instillations. Dilutions were 1:400 for primary and 1:40 000 for challenge. *Means arc significantly different (P < 0.05) when aged primary values are compared with young primary values.

198

(Fig. 2). Following challenge antigen instillation, lavage fluid anti-KLH IgA and IgG antibody responses remained significantly lower in the RCL of aged dogs than in young dogs (Fig. 2). No significant differences between aged and young dogs were found in the RIL (control lobe; O.D. values < 0.2 for RIL). In vitro tissue cell responses

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Fig. 2. Lavage fluid anti-KLH IgA, IgG, IgM antibody responses (~ a- S.E.M.) in the RCL (KLH instilled) and RIL (control = saline instilled) lung lobes of aged and young dogs following primary and challenge antigen instillations. IgA, IgG dilution 1:80 (primary), 1:4000 (challenge); IgM hl0. *Means are significantly different (P < 0.05) when aged primary values are compared with young primary values, or when aged challenge values are compared with young challenge values.

199 of the RCL of aged dogs were significantly lower than those of young dogs following primary antigen instillation, while only IgA and IgG were lower in aged dogs after antigen challenge. Cells from the lung tissue of the RCL of aged dogs produced significantly less anti-KLH IgA and IgM after primary antigen instillation and significantly less IgA, IgG and IgM after antigen challenge. In vitro anti-KLH IgM antibody responses by cells from lavage fluid and lung tissue of the RIL were significantly lower in aged dogs than in young dogs only after primary antigen instillation (Fig. 3). In vitro anti-SRBC IgM antibody responses were not measured because of insufficient numbers of cells. No significant differences were found when comparing in vitro anti-KLH or antiSRBC antibody production by peripheral blood lymphocytes of aged and young dogs (data not shown). A low level of anti-KLH antibody production was observed in the spleen and TBLN and no antibody production was found in distant lymph nodes (mesenteric and popliteal). After primary antigen instillation, production of anti-KLH IgG and

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Fig. 3. Anti-KLHIgA, IgG, IgM antibodyproductionfromin vitro cell culturesof lymphocytesfrom lavageand lungtissue. Comparisonof aged and youngdog anti-KLHantibodyresponses~ 4- S.E.M.) followingprimaryand challengeantigeninstillations.IgA, IgG dilution h 10 (primary), 1:80(challenge); IgM no dilution.*Meansare significantlydifferent(P < 0.05)whenagedprimaryvaluesare compared with youngprimaryvalues, or when aged challengevalues are comparedwith youngchallengevalues.

200

TABLE I SPLENIC ANTI-KLH A N T I B O D Y P R O D U C T I O N BY A G E D A N D Y O U N G DOGS

Antibodies to KLH in spleent lgA

lgG

IgM

0.26 4- 0.20 1.60 4- 0.40

0.06 4- 0.06* 1.91 4- 0.28

0.07 4- 0.07* 0.45 4- 0.10

0.34 4- 0.13 0.95 4- 0.50

1.02 4- 0.45* 1.71 4- 0.32

0.83 4- 0.46* 0.25 4- 0.06

Aged Primary Challenge

Young Primary Challenge

*Means are significantly different (P < 0.05) when aged primary values are compared with young primary values, or when aged challenge values are compared with young challenge values. All values are means 4- S.E.M. tStandardized O.D. readings.

IgM by spleen cells from young dogs was significantly higher (P < 0.05) than in aged dogs, while there were no significant age-related differences in IgA production (Table I). There were no significant differences in anti-KLH IgG, IgM, or IgA production between aged and young dogs following antigen challenge. Differential cell counts In both the RCL and the LCL, the numbers of lymphocytes, neutrophils and macrophages were significantly higher (P < 0.05) in young than in aged dogs after primary antigen instillation (Table II). The numbers of lymphocytes and macrophages

TABLE II D I F F E R E N T I A L CELL COUNTS IN A G E D A N D Y O U N G DOGS

LCL Lymph

RCL PM N

Mac

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PM N

Mac

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2.1 4- 1.0" 11.6 4- 4.8*

0.8 4- 0.6* 3.8 4- 2.0

16.0 4- 4.0* 25.5 4- 5.1"

1.7 4- 0.7* 8.3 4- 2.6*

0.7 ± 0.2* 1.2 4- 0.5

14.3 4- 2.3* 15.5 4- 3.6*

14.2 4- 7.8* 37.0 4- 9.0*

15.2 4- 4.2* 8.3 4- 2.8

37.4 4- 5.1' 54.1 4- 9.3*

6.4 4- 1.9" 20.0 4- 3.6*

10.0 4- 3.8* 3.4 4- 1.8

39.4 4- 7.6* 42.8 4- 8.8*

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*Means are significantly different (P < 0.05) when aged primary values are compared with young primary values, or when aged challenge values are compared with young challenge values. All values are means ± S.E.M. aTotal cells lavaged from LCL and RCL in millions.

201

were significantly lower (P < 0.05) in aged than in young dogs in both the LCL and RCL after antigen challenge, but there were no significant differences in neutrophil numbers (Table II). There were also no significant differences between aged and young dogs in RIL cell numbers at either primary or challenge timepoints (data not shown). Serum albumin, lavage fluid albumin and total protein levels Aged dogs had lower (P < 0.05) amounts of serum albumin than young dogs after both primary and challenge antigen instillation (Table III). Albumin levels in both the LCL and RCL were significantly lower (P < 0.05) in aged than young dogs after primary antigen instillation (Table III). There were no significant differences after antigen challenge. Amounts of total protein were significantly higher (P < 0.05) in young than in aged dogs in the LCL and RCL, but not in the RIL, after primary antigen exposure (Table III). There were no significant differences between aged and young dogs in total protein in any lung lobe after antigen challenge. Immune memory responses The abilities of aged and young dogs to form a localized anti-KLH immune response, or immune memory response, in the RCL following antigen challenge was compared by measuring anti-KLH antibody responses in the LCL (SRBC-instilled). The anti-KLH antibody in the LCL following challenge antigen instillation is produced by cells that enter the LCL from the systemic circulation (see Discussion). By comparing anti-KLH antibody responses in the LCL with anti-KLH responses in the RCL, it was possible to compare localized anti-KLH antibody production (immune memory) in the RCL between aged and young dogs. TABLE III SERUM A N D L ~ V A G E ALBUMIN A N D LAVAGE TOTAL PROTEIN IN Y O U N G A N D AGED DOGS

Serum albumin a

Lavage albumin a

Lavage total protein a

LCL

RCL

RIL

LCL

RCL

AlL

Aged Primary Challenge

4 8 0 ± 17" 490 ± I1"

23 ± 1" 41 ± 8

2 4 ± 1" 30 ± 3

24± 1 23 ± 1

1 2 4 ± 20* 643 ± 243

181 ± 22* 444 ± 119

114±25 112 ± 23

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542 ± 10" 532 ± 13"

58 q- 9* 47 ± 5

38 ± 7* 34 ± 2

23 a- 1 22 ± 1

786 ± 220* 872 ± 198

437 a- 149" 586 ± 115

88 ± 10 50 ± 12

*Means are significantly different (P < 0.05) when aged primary values are compared with young primary values, or when aged challenge values are compared with young challenge values. All values are means ± S.E.M. aValues are t~g/ml.

202

Aged dogs had a decreased ability to form a localized immune memory response compared to young dogs. A comparison of the anti-KLH responses in the lavage fluid from the LCL and RCL showed no significant immune memory responses in aged dogs (Fig. 4). However, evaluations of lavage fluids from the RCL and LCL of young dogs showed memory responses for both IgA and IgG (Fig. 4). The only 2.0

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203

significant immune memory response for aged dogs was the production of anti-KLH IgA and IgM by cells from lung tissue (Fig. 5). Significant immune memory responses were observed in young dogs for anti-KLH IgA, IgG and IgM production by lavage and lung tissue cells (Fig. 5). 20 -

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Fig. 5. Immune memory responses in aged and young dogs. Comparison of anti-KLH antibody production by cells from lavage fluid and tissues. The LCL was challenged with SRBC to induce inflammation and cells producing anti-KLH antibody in this lobe were recruited from blood. Antibody production by cells from lavage fluid and tissues from the RCL represent both recruitment from blood and local proliferation.

204 Anti-SRBC responses

Anti-SRBC antibody responses followed the same trends as the anti-KLH data for serum, lavage fluid and in vitro tissue responses for primary and secondary antigen instillation. Immune memory comparison of anti-SRBC in the LCL and RCL was also similar to the anti-KLH data. Aged dogs had virtually no measurable differences in anti-SRBC antibody responses in the LCL compared to the RCL. Because of differences in antigenicity, anti-SRBC responses were at a lower level, however, than the anti-KLH for both aged and young dogs (data not shown). DISCUSSION

Our results show that antigen-specific antibody in serum was significantly lower in aged dogs following a primary lung immunization, but not after antigen challenge. These findings are similar to results in other studies in which age comparisons were made [11,12,24]. However, in the lung, aged dogs had significantly lower antigenspecific antibody responses after primary immunization and antigen challenge. This was true for lavage fluid, lavage cell and lung tissue cell antibody responses. Localized immune memory responses following challenge antigen instillation, were also significantly lower in aged than in young dogs. It has been suggested by other researchers that the mucosal immune system (primarily gut-associated immune responses) is resistant to the age-related decline observed in the systemic immune response [1,3-5,24]. The lung and its associated lymph nodes are considered a part of the mucosal immune system. Our study is the first to demonstrate that localized lung immune responses are significantly depressed in aged dogs, even following antigen challenge. The ability to generate localized immune responses in the lungs may have an important role in the prevention of pulmonary infections or degenerative disease processes. The primary immune response to antigen deposition in the lung involves the translocation of antigen to the TBLN where a primary immune response is produced [15,20,22]. Antigen-specific antibody forming cells (AFC) and antibody produced by this primary immune response are then released into the circulation where they are recruited back into the immunized lung lobe. Bice et al. showed that the accumulation of AFC into immunized lung lobes after a primary immunization is not antigenspecific [16]. It was concluded that the primary immune response was due to the accumulation of AFC from blood in a non-specific manner secondary to the inflammation resulting from antigen deposition. Additional data show that after a primary immune response is established, the secondary immune response is generated by local clonal expansion of antigen-specific lymphocytes within the lung [14,17]. It is reasonable to assume, based on the above findings, that when two different antigens are first instilled into different lung lobes, AFC will be recruited into both lung lobes in a non-specific manner. However, following a repeated antigen instillation into the lung lobes, the antibody responses at each site of antigen instillation

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become antigen-specific, with antibody responses in each antigen-challenged lung lobe resulting from antigen-specific memory cells. Any antibody responses to the other antigen will be primarily due to recruitment of systemic AFC into the lung and not to antigen stimulation of immune memory cells. Therefore, by comparing antiKLH antibody responses in the RCL (KLH-instilled lung lobe) to anti-KLH antibody responses in the LCL (SRBC instilled lung lobe), we could measure the ability of aged and young dogs to develop immune memory within an antigen-challenged lung lobe. Our observation that aged dogs did not develop a normal localized immune memory response in lung lobes exposed to either KLH or SRBC is an important finding. The reasons for this deficit in immune memory are unclear, but the possibilities are numerous. It may be that there is a decline in resident memory B cells within the lung with increasing age or that the ability of clones of antigen-specific B cells to expand is suppressed, as suggested in several publications [2,3,25]. Whatever the reason, the lack of localized immune memory in the lung lobes of aged dogs portends the likelihood of increased infections or degenerative diseases. The lack of antibody response in the TBLN is most likely due to the time of sampling (i.e., late in the time-course for peak lymph node response). There was also a marked difference in the physical appearance of the lymph nodes between aged and young dogs. Aged dog TBLN were small and atrophied, while those of young dogs were large and reactive (based on cytology), except for the one 17-year-old dog whose TBLN were of good size and reactive. The one 17-year-old dog used in this study was an outlier not only with respect to his age, but also with respect to his antigen-specific antibody responses (data not shown). Many of his immune responses in serum, lavage and tissue were equivalent to those of a 2- to 3-year-old dog. This supports findings that healthy, long-lived humans have active immune responses which may contribute to their long lives and good health status [26,27]. The data from this dog were included in all comparisons of aged dogs. There are several mechanisms that may be responsible for the decline in systemic and localized immune responsiveness (reviewed in Refs. 1-5) of aged animals, including diminished activity of T helper cells and their factors as well as problems relating to antigen handling and down-regulation of immune responses. The possible role of anti-idiotypic antibody in the down-regulation of antigen-specific antibody responses with age has been investigated [28,29]. Most of the research on this phenomenon has been done in mice, but the fact that most aged humans and other animals have higher levels of circulating, low-affinity antibody supports these findings [2,5,25]; however, we did not find significant differences in circulating total IgG or IgM antibody between the aged and young dogs in this study. Whatever the mechanisms responsible for the decline of antibody responses with age, the inability of aged dogs to develop a localized immune response in the lungs may help to explain the increased incidence of pulmonary infections and degenerative disease processes seen in aged humans as well as dogs and other animals.

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We conclude from this study that aging of the dog is associated with suppressed pulmonary immune responses and a marked suppression of pulmonary immune memory, in addition to reduced systemic immune responses. This study also shows that there is a close correlation between antibody production by lavage cells and antibody production by lung tissue cells. ACKNOWLEDGEMENTS

The authors wish to thank A.J. Williams, C.I. Kenyon and D.S. Swafford for their technical assistance and M.A. Weinhold, S. Wilson and the Animal Care staff for their assistance and care of the animals involved in this study. We thank Dr. David Weissman (Veterans Hospital, Albuquerque, NM) for his gift of KLH used in this study. This research was supported by the U.S. Department of Energy under Contract DE AC04-76EV01013. Animals were cared for in accordance with the NIH guide for the care and use of laboratory animals, in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. REFERENCES 1 A.W. Wade and M.R. Szewczuk, Aging, idiotypic repertoire shifts and compartmentalization of the mucosal-associated lymphoid system. Adv. Immunol., 36 (1984) 143-188. 2 T. Makinodan and M.M. Kay, Age influence on the immune system. Adv. Immunol., 29 (1980) 287-330. 3 M.L. Thoman and W.O. Weigle, The cellular and subcellular bases of immunosenescence. Adv. Immunol., 46 (1989) 221-261• 4 M.R. Szewczuk and A.W. Wade, Aging and the mucosal-associated lymphoid system. Ann. N Y Acad Sci., 409 (1983) 333-344. 5 A.W. Wade, J. Green-Johnson and M.R. Szewczuk, Functional changes in systemic and mucosal lymphocyte repertoires with age: an update review• Aging: Immunol. Infect. Dis., 1 (1988) 65-97. 6 T. Makinodan, S.J, James, T. Inamizu and M.-P. Chang, Immunologic basis for susceptibility to infection in the aged. Gerontology, 30 (1984) 279-289. 7 R.L. Walford, Immunology and aging. Am. Soc. Clin. Pathol., 2 (1980) 247-253. 8 M.S. Finkelstein, Unusual features of aging. Geriatrics, 37 (1982) 65-78• 9 M. Rothstein, Biochemical studies of aging. Chem. Eng. News August, 11 (1986) 26-39• 10 J.P. Phair, Aging and infection: a review. J. Chron• Dis, 32 (1979) 535-540. 11 A.L. Esposito and J.E. Pennington, Effects of aging on antibacterial mechanisms in experimental pneumonia. Am. Rev. Respir. Dis., 128 (1983) 662-667. 12 D.E. Bice and B.A. Muggenburg, Effect of age on antibody responses after lung immunization. Am. Rev. Respir. Dis., 132 (1985) 661-665. 13 D.E. Bice and G.M. Shopp, Antibody responses after lung immunization. Exp. Lung Res., 14 (1988) 133-155. 14 D.E. Bice and B.A. Muggenburg, Localized immune memory responses in the lung. Am. Rev. Respir. Dis., 138 (1988) 565-571. 15 • H.B. Kaltreider and F.N. Turner, Appearance of antibody-forming cells in lymphocytes from the lower respiratory tract of the dog following intrapulmonary or intravenous immunization with sheep erythrocytes. Am. Rev. Respir. Dis., 113 (1976) 613-617. 16 D.E. Bice, M.A. Degen, D.L. Harris and B.A Muggenburg, Recruitment of antibody forming cells in the lung after local immunization is nonspecific. Am• Rev. Respir. Dis., 126 (1982) 635-639.

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