INTRODUCTION

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Under normal conditions, a small but definite amount of protein crosses the alveolar epithelium into the pulmonary and systemic circulations (1). However, the alveolar epithelial permeability to protein has been shown to increase after inflammatory changes in the lungs (7). The way the lung handles inhaled proteins under normal conditions and in an immunized host may have consequences for the levels of circulating antigens and the development of immunologic and inflammatory processes. It has previously been suggested that immunization reduces the passage of soluble antigens across the epithelia in the respiratory tract (10). Similar observations have been made in the gastrointestinal tract, where in addition the permeability to an unrelated bystander protein was increased (14, 15).

The aims of this study were (1) to characterize the effects of specific immunization on alveolar epithelial permeability to protein, (2) to investigate whether local lung immunization would affect alveolar epithelial protein permeability, and (3) to study if an inflammation caused by the antigen-antibody reaction would affect the barrier permeabilities to protein. The passages across the alveolar epithelium of both antigen and a bystander protein that was closely related to the antigen in size and other physiochemical properties but not immunologically cross-reactive were studied in immunized rats.

	METHODS

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Animals

Male Sprague Dawley rats (n = 70) weighing 300 to 350 g (ALAB, Sollentuna, Sweden) were kept at a 12:12 h day-night rhythm at 20 to 22° C. The rats were fed with a standard rat chow (ALAB) and had water ad libitum. The experiments were approved by the Ethical Review Committee on Animal Experiments at Lund University.

Immunization

All rats were immunized 1 mo before start of the experiments and a booster immunization was given 14 d before start of the experiments. For intradermal immunization, a solution of 5 mg/ml bovine serum albumin (BSA) in 0.9% NaCl emulsified with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI) was prepared for the primary immunizations of the rats. For the booster immunization, BSA was emulsified with Freund's incomplete adjuvant (Difco Laboratories) as the adjuvant substance. The intradermal immunization was done by injecting 0.2 ml/animal of the immunization solution into two areas on each animal's back.

For intrapulmonary immunization, a solution of 5 mg/ml BSA in 0.9% NaCl with and without addition of 5 mg/ml dextran 70,000 (Pharmacia-Upjohn, Uppsala, Sweden) as an adjuvant substance was used. Dextran has been shown in rats to cause pulmonary inflammation with little or no effects on alveolar epithelial permeability, but generating a significant influx of inflammatory cells (7). Intrapulmonary immunization was done by intratracheal instillation (see below and reference 2) of the lung immunization solution (1 ml/kg body weight).

Antigen-Bystander Solutions

The antigen-bystander solution was prepared by dissolving 5 mg/ml BSA, (Sigma Chemical Co., St. Louis, MO) and 5 mg/ml human serum albumin (HSA; Sigma Chemical) in 0.9% NaCl.

General Experimental Protocol

Immediately before the intratracheal instillation, a 2-ml blood sample was taken by heart puncture under ether anesthesia. The rats were placed on a slanted board (20 degrees from vertical) hanging by their upper incisors and the antigen-bystander solution was delivered via the mouth to the trachea using a modified syringe needle in a volume of 1 ml/kg body weight (2). After instillation, the rats were allowed to recover in their cages where they remained during the 16-h experimental period.

At the end of the experimental period, a blood sample was taken by heart puncture. The blood was centrifuged and the serum was stored frozen at 20° C until analysis. Directly after the exsanguination by heart puncture, a bronchoalveolar lavage was done (2). The lavage was centrifuged at 3,000 × g and the supernatant was frozen at 20° C for measurement of total protein and antibody titer. The pelleted cells were resuspended in 1 ml 0.9% NaCl and counted for total cell number.

Specific Protocols

Group 1. Nonimmunized control rats (n = 10). Half of the rats in this group were injected intradermally with 0.9% NaCl and half were instilled intratracheally with 0.9% NaCl.

Group 2. Intradermal immunization (n = 30). These rats were immunized by intradermal immunization on two areas of each rat's back with BSA plus Freund's complete adjuvant. The booster was done 14 d later with BSA plus Freund's incomplete adjuvant.

Group 3. Intrapulmonary immunization with (n = 17) and without adjuvant (n = 13). These rats were immunized by an intratracheal instillation of 5 mg/ml BSA with or without 5 mg/ml dextran as adjuvant and inflammation-inducing substance.

Analyses

Measurements of immunoreactive antigen (BSA) and bystander protein (HSA) concentrations. BSA and HSA levels in serum and lavage fluid were measured by electroimmunoassay (16), using rabbit antibodies to BSA and HSA (Dakopatts AB, Hägersten, Sweden). Any degradation of BSA and HSA was estimated by crossed immunoelectrophoresis (17). Areas under the respective precipitation peaks were used to estimate the relative amounts of intact or degraded BSA or HSA.

Lavage analyses. Total protein in the bronchoalveolar lavage fluid was measured using the technique of Lowry and colleagues (18), modified for use with microtiter plates.

Measurement of specific antibody titers to BSA. Antibody titers in blood serum and lavage fluid were measured using a specific ELISA procedure (19) using rabbit antirat IgG as primary antibody (Dakopatts AB) and phosphatase-conjugated swine antirabbit IgG as secondary antibody (Dakopatts AB).

All the data are summarized and presented as mean ± SD. The data were analyzed by one-way analysis of variance (ANOVA) with Student-Newman-Keuls post hoc test, or Student's t test, when appropriate. Statistical significance was set as p < 0.05.

	RESULTS

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Specific Antibody Titers Following Immunization

We measured serum and bronchoalveolar lavage titers of specific IgG antibodies to the antigen BSA after intradermal and intrapulmonary immunization. Serum titers of specific IgG (anti-BSA) were generally higher in intradermally immunized rats than in intrapulmonary immunized rats, but no specific IgG was detected in nonimmunized control rats (Figure 1, top panel ). Specific IgG serum titers were higher in rats intrapulmonary immunized with BSA together with dextran as adjuvant than with only BSA (Figure 1, top panel ). Specific IgG was detected only in lavage fluid from intradermally immunized rats, though the titers were lower than in serum (Figure 1, top panel ).

View larger version (K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 1.   Systemic and local antibody titers (top panel ) and alveolar epithelial permeability to protein (bottom panel ) in control rats and in rats immunized against BSA either intradermally or intrapulmonary. (Top panel ) Effect of different schemes of immunization on specific IgG (anti-BSA) titers in serum (closed bars) and in bronchoalveolar lavage fluid (open bars) 16 h after instillation of the antigen (BSA) and a bystander protein (HSA). Intradermal immunization resulted in significantly higher specific IgG titers than did intrapulmonary immunization. Specific IgG titers were seen only in the bronchoalveolar lavage fluid of intradermally immunized rats. Intrapulmonary immunization with dextran resulted in higher specific IgG serum levels than intrapulmonary immunization with BSA alone. (Bottom panel ) Serum levels of the antigen BSA (closed bars) and the bystander protein HSA (open bars) 16 h after instillation of the antigen/bystander solution in control rats, intradermally immunized rats, and in intrapulmonary immunized rats with and without dextran as adjuvant substance. There were no detectable amounts of BSA after intradermal immunization in the serum. However, the serum levels of HSA were significantly higher in rats that were immunized intradermally than in nonimmunized control rats. No changes in the serum levels of the antigen or bystander protein as compared with nonimmunized control rats were observed in the intrapulmonary immunized rats. Data are presented as means ± SD. Statistical evaluation was done with one-way ANOVA with Student-Newman-Keuls test as post hoc. *p < 0.05 compared with control rats; p < 0.05 compared with intrapulmonary immunization with BSA alone; p < 0.05 compared with intrapulmonary immunization with BSA and dextran.

Alveolar Epithelial Permeability to Antigen and Bystander Protein

Control rats. In the nonimmunized control rats intratracheally instilled with the antigen and bystander protein in equal amounts, serum levels of BSA and HSA were similar 16 h after instillation (Figure 1, bottom panel ).

Intradermal immunization. In the rats intradermally immunized against BSA, immunoreactive BSA was undetectable in serum (Figure 1, bottom panel ). In contrast, serum levels of the bystander protein HSA were markedly increased compared with those in control rats (Figure 1, bottom panel ).

Intrapulmonary immunization. In rats immunized with BSA intratracheally, either alone or coadministrated with dextran, alveolar epithelial permeabilities to both the antigen BSA and bystander protein HSA were unchanged compared with those in control rats (Figure 1, bottom panel ).

Inflammatory Parameters Measured in the Lung

We also lavaged the air spaces of the lungs of rats for protein concentration and cell influx immediately after the permeability experiments were done. Total protein levels in lavage fluid were used to estimate endothelial-epithelial injury caused by antigen provocation. An earlier intradermal immunization resulted in an increased protein amount in the lavage fluid as compared with nonimmunized control rats (Table 1). Intrapulmonary immunization, however, resulted in an increased endothelial-epithelial protein leak only when dextran was coinstilled as adjuvant substance (Table 1).

Total cell number in the air spaces was unchanged in intradermally immunized rats (Table 1). In intrapulmonary immunized rats, total cell number was increased twofold compared with control rats independently of use of dextran as adjuvant substance (Table 1). No differential cell count was carried out on these bronchoalveolar lavage samples.

	DISCUSSION

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There are two principal findings in this study. First, presence of specific IgG (anti-BSA) in the bronchoalveolar lavage was correlated with an increased alveolar epithelial permeability to protein. These antibodies may have leaked out into the alveolar spaces after antigen instillation or they may have been there from the start. We could not distinguish between these possibilities since we did the bronchoalveolar lavage after antigen instillation. Second, intradermal immunization was more efficient in producing a high serum titer of specific IgG (anti-BSA) than was intrapulmonary immunization. These findings suggest that the presence of high levels of circulating specific antibodies may influence alveolar epithelial permeability to protein. This finding may have implications for sensitive asthmatics since an increased nonspecific permeability to potential antigens may result in development of further allergies, nonrelated to the original disease.

It has been suggested in earlier studies that a preceding immunization may reduce or even totally inhibit the passage of protein antigens from the alveolar spaces to the systemic circulation (10, 20). However, these studies used only the specific antigen for estimating the permeability to protein, and much of the effects may be attributed to an increased degradation of the protein antigen or a reduced penetration across the alveolar epithelium because of the increased molecular size when antigen and antibody form immune complexes. We used HSA as a bystander protein that was similar in most characteristics, e.g., size, as the antigen BSA and consequently HSA crossed the alveolar epithelium to the same extent as BSA in nonimmunized rats. A changed serum concentration of HSA after intratracheal instillation in immunized rats was considered evidence for an altered alveolar epithelial permeability to protein. Thus, in rats intradermally immunized with BSA, after instillation, the HSA serum levels were significantly increased threefold compared with those in the control rats. This suggests that the general nonspecific alveolar epithelial permeability to protein was increased after immunization when antigen is again present in the air spaces of the lung.

The fact that we could not measure the protein antigen BSA in the serum after the intradermal immunization could have several explanations, but it was probably due to the high levels of circulating specific antibodies that had complex bound BSA. Our assay antibodies probably recognized the same antigenic determinant and therefore we could not detect BSA in the serum of these rats. Alternatively, the presence of specific IgG (anti-BSA) in the alveolar space in these rats bound the BSA. Because of this binding, the size of the molecule drastically increased and its permeability decreased; it has been demonstrated that alveolar epithelial permeability to protein is dependent on the molecule's size (2). This has previously also been suggested to happen after instillation of antigen into immunized lungs (13).

In contrast to intradermal immunization, intrapulmonary immunization did not change alveolar epithelial permeability to either the antigen or the bystander protein, even when it was done with dextran as the adjuvant substance. This may be explained by two possibilities. First, the relatively low serum titers of specific IgG (anti-BSA) were not enough to create an inflammatory response. Second, absence of specific IgG (anti-BSA) in the air spaces, as reflected in the bronchoalveolar lavage fluid, in intrapulmonary immunized rats may not have resulted in immune complex formation and therefore no activation of complement. However, influx of inflammatory cells, as seen by increased cell numbers in the bronchoalveolar lavage, was significant. Also, endothelial-epithelial leak of protein into the air spaces was elevated, especially if dextran had been given at the time of immunization. This low increase in endothelial-epithelial permeability to protein could, however, have been caused by accumulation of antibodies specific to the antigen BSA in the air spaces. It is possible that the low systemic antibody titers as well as the limited inflammatory response after antigen instillation following intrapulmonary immunization was due to local immune responses that never reached the systemic circulation. A limited weak immune response was also found in a study of intratracheal immunization with sheep erythrocytes in mice (21). In fact, it was an absolute requirement that the lungs were reexposed to the antigen in order to get a good immune response via the lung immunization. The main function of secretory mucosal antibodies is in cooperation with innate defense mechanisms to execute immune exclusion (22). Therefore, entrance of antigen into the systemic circulation may have been limited and did not cause a major circulatory antibody response or sensitization via the lung is restricted.

It has been demonstrated that after exposure of sensitive asthmatics to allergen, significant levels of protein have been observed in bronchoalveolar lavage fluid (23). Therefore, this experimental study and the clinical study by Fick and colleagues (23) suggest that allergen exposure to sensitized subjects causes an acute increase in both bronchovascular permeability to serum proteins and an acute increase in alveolar epithelial permeability to protein.