Interaction between environmental pollutants and respiratory infections.

The major aspects that must be considered in studies of the health effects of environmental pollutants are: the direct damage due to the exposure, the role of pre-existing disease, and effects of the exposure on the response to secondary stresses. In experimental studies at concentrations of air pollutants found in urban environments frank toxicological responses are rarely observed. However, exposure to a secondary stress, i.e. respiratory challenge with infectious bacteria, can exacerbate the response of the experimental host. Changes in the resistance to respiratory infections provide a highly sensitive experimental animal model system, which is increasingly used in studies of health effects of air pollutants. This model indicates the impairment of the basic defense mechanisms of the respiratory system by the combined exposure to low concentrations of pollutants and the superimposed bacterial infection. Changes in the resistance to respiratory infections were studied in various species of laboratory animals. S. pyogenes and K. pneumoniae are the bacteria of choice to induce the pulmonary infection. Included in the studies are short-term single and multiple exposures as well as long-term exposures to gaseous pollutants such as O3 and NO2 and particulate pollutants such as sulfates and nitrates. Changes in the resistance are measured as excess mortalities and reduced survival time as compared to those in infected animals not exposed to the pollutants. Other parameters measured ranged from changes in the immune response to changes in retention rates of bacteria in lungs.


Introduction
Several papers presented at this symposium have dealt with the mechanisms which are active in protecting the respiratory tract against damage due to inhalation of airborne contaminants. The mechanisms include the mucociliary system, phagocytic activity of alveolar macrophages, and the immunity of the respiratory tract. These mechanisms are indeed the same which play a significant role in protecting a host against respiratory infections. Thus, the effects of air pollutants on the respiratory tract are of special importance in relation to respiratory infections.
Health effects studies of air pollutants have been usually concerned with the causal association between a single pollutant and a disease state. However, it is well known that frequently more than one factor is responsible for the occurrence of natural diseases. Therefore, multiple causalities must be considered in the assessment of biological effects of air pollutants. One such interaction is depicted by the animal model system which reflects the exacerbation of bacterial respiratory infections by inhalation of air pollutants. Indeed, changes in the resistance to respiratory infections provide a highly sensitive experimental model which is with increasing frequency used in studies of health effects of air pollutants.
The relationship between exposure to air pollutants and the resistance to respiratory infections has been investigated in our laboratories since 1955. The gaseous pollutants included in these studies were ozone, nitrogen dioxide, sulfur dioxide, and peroxyacetyl nitrate. Particulate pollutants included sulfuric acid, various sulfates and nitrates, and oxides of platinum, palladium, manganese, and nickel. The studies were designed to determine the effects of single and multiple exposures to a single pollutant or to pollutant mixtures.

Materials and Methods
The experimental protocol used in the studies calls for exposure of a group of animals, usually 6 to 8-week old mice, to a pollutant and another group to clean air. After the exposure the two groups of mice are combined and within less than 1 hr challenged by the respiratory route with an aerosol of the infectious bacteria, either Streptococcus pyogenes or Klebsiella pneumoniae. The challenge usually lasts for 10 to 15 min and results in the deposition of approximately 750 to 1500 viable bacteria per lung. After the challenge the mice are kept in an isolated clean-air room for 14 days observation period. The changes in resistance to the infection are usually measured by two parameters, namely, changes in mortality rates and in survival time when compared to those seen in infected mice exposed to filtered air. In addition to mortality and survival, several other parameters are measured. They include clearance rate of inhaled viable bacteria from the lungs, damage to the respiratory tract tissue as observed by conventional histopathological and scanning electron microscopic examination, development of lung edema, activity and function of alveolar macrophages, humoral and cell-mediated immune responses and various clinical pathology measurements. To determine the statistical significance of the differences in responses between animals exposed to the pollutants and those exposed to clean air the chi-square analysis, Student's t-test, analysis of variance and linear regression analysis were used, as appropriate. The details of experimental methods used for preparation of the environmental atmospheres, monitoring of the pollutants, infectious challenge with the bacterial aerosols and the assay of the various health parameters were described in previous publications (1)(2)(3). Figure 1 shows mortalities resulting from a single 3-hr exposure of mice to either nitrogen dioxide or ozone and challenge with Streptococcus aerosol (2). Statistically significant increases (p s 0.05) in actual mortality are seen after the single exposure to either 3.76 mg/m3 N02 or 0.2 mg/m3 03. These data indicated the high sensitivity of this assay system, whereby a significant physiological response can be attained at concentrations of air pollutants frequently found in urban environments. The mortality rates of mice exposed to ozone are in a close agreement with those reported by Coffin et al. (4). In his work the death rate in control mice was 10.6%, in those exposed to 0.14 mg/m3 0320.0%, to 0.2 mg/m3 0335.0 and to 0.98 mg/m3 0380.0%. Thus the reproducibility of this animal model system among two different laboratories and during two different time periods is apparent.

Effects of Gaseous Pollutants
In an urban environment the concentrations of air pollutants vary considerably with their rate of emission and meteorological conditions. This frequently results in presence of low baseline concentrations of a pollutant with superimposed short-duration peaks of higher concentration of the same pollutant.  dioxide. Subsequent sunlight irradiation of nitrogen dioxide results in increase in concentrations of ozone. Human populations therefore can be exposed over extended periods of time to low concentrations of N02 or to low concentrations of this pollutant with superimposed peaks of elevated concentrations of N02 and O0 mixtures. Figure 2 shows the effects of continuous 24 hr/day, 7 day/week exposure to 0.94 mg/m3 (0.5 ppm) or 2.82 mg/m3 (1.5 ppm) N02 on the susceptibility to K. pneumoniae infection (5). The increased mortality rates in mice exposed to 0.94 mg/m3 N02 for 3 months or longer, and to 2.82 mg/m3 N02 for 8 hr or longer were significant (p -0.05). Excess mortality was also present in mice exposed to nitrogen dioxide for the shorter time periods, but the differences were not statistically significant.
To determine the effects of inhalation of nitrogen dioxide and ozone mixtures, groups of mice were exposed for 3 hr to various concentrations of each pollutant, a mixture containing the same concentration of the two pollutants or to filtered air. The four groups were then simultaneously challenged with Streptococcus aerosol. The differences between mortality among mice exposed to the pollutants and the corresponding control mice, challenged with the bacterial aerosol but exposed to filtered air are shown in Table 1 (2). The data indicate that the effects of the 3-hr exposure to the N02 and 03 mixture were additive. In most instances the excess mortalities were equivalent or somewhat higher than the sum induced by exposure to each individual pollutant.
To further simulate the realistic condition of urban population exposure, groups of mice were exposed inhalation of the infectious bacteria, groups of mice continuously 24 hr/day (7 day/week) to a backwere exposed to the pollutants before and for the ground concentration of 0.19 mg/m3 N02 with 14-day period following the infectious challenge. Resuperimposed 3-hr daily peaks (5 days/week) of sults shown in Figure 3 indicate that the combination either 0.94 mg/m3 N02 or a mixture consisting of of extended exposure to the pollutants and continu-0.94 mg/m3 N02 and 0.20 mg/m3 03. Inasmuch as ation of the exposure after the respiratory challenge under true environmental conditions exposure to the with Streptococcus aerosol was most deleterious. pollutants can be expected also to occur after the Upon such an exposure sequence increased mortal- Environmental Health Perspectives ity and shortened survival time (see Fig. 4) were seen in almost all exposure regimens. Moreover, these changes in resistance appeared sooner than when mice were exposed to clean air after the challenge. Another important factor appeared to be the stress of repeated daily exposure to the N02 and 03 mixture. Irrespective whether or not the mice were reexposed to the pollutants or kept in clean air after the infectious challenge, increased mortality was seen in this group after 2 months or longer exposures.
The reduced mortality rates during the first 3 months of continuous exposure to 0.1 ppm N02 with the superimposed daily 3-hr peaks of either nitrogen dioxide and ozone mixture or 0.5 ppm N02 could possibly represent initial adaptation or a protective effect of the continuous exposure to nitrogen dioxide. This response occurred only after withdrawal from the polluted atmosphere and appeared to be negated by continuation of the exposure to the pollutants either for an additional 3 months or for the 14-day period after the infectious challenge.  bSignificant change compared to infected mice exposed to filtered air (p < 0.05).
The third gaseous pollutant included in the studies Effects of Particulate Pollutants was sulfur dioxide (S02). These experiments encompassed single and multiple 3-hr exposures as Although a considerable amount of information is well as continuous 24 hr/day, 7 days/week exposures now available on the effects of photochemical oxito 13.1 mg/m3 (5 ppm) S02. As seen in Table 2, dant pollutants on the resistance to infection there is exposure to S02 had no effect on the resistance to a paucity of data pertaining to the effects of particustreptococcal pneumonia as measured by changes in late pollutants, especially sulfates or nitrates. The mortality rates or survival time. The one statistically tables and figures summarize results more recently significant increase in mortality seen after 2 months obtained in our laboratories. The results further of continuous exposure appears to be of no practical confirm the utility of this experimental animal model or true importance, since all the other values obsystem for studies of air pollutants as well as entained in these exposures did not indicate any vironmental contaminants of importance to occupachanges in resistance. Other health effect parame-tional health. ters measured during these studies, such as viability Changes in susceptibility to streptococcal pneuand phagocytic activity of macrophages, develop-monia caused by a single 3-hr exposure to zinc sulment of lung edema, body weights, rectal tempera-fate, zinc ammonium sulfate, and ammonium sulfate ture, and SEM examination of lungs, trachea, and are shown in Table 3. Inhalation of 1.2 mg/m3 or nasal cavities, also did not show any changes which more zinc sulfate or 2.1 mg/m3 or more zinc amcould be ascribed to the exposure. monium sulfate followed by a respiratory challenge dSignificant change from infected mice exposed to filtered air (p S 0.05).
Environmental Health Perspectives with airborne Streptococcus resulted in significant increases in mortality and in reduced survival time in mice. Exposure to ammonium sulfate aerosol in concentrations up to 5.3 mg/m3 had no effect on susceptibility to the infection (3).
The changes in resistance to infection appeared to be related to the zinc ion present in the sulfate complex. The lesser effect of zinc ammonium sulfate relative to zinc sulfate in enhancing the severity of The data shown in Figures 5 and 6 and Table 4 appear to confirm these assumptions. Figure 5 shows the least square lines, and the corresponding 95% confidence limit resulting from linear regression analysis of excess mortalities produced by a 3-hr  aSignificant change from infected mice exposed to filtered air (p S 0.05).
inhalation of zinc sulfate and zinc nitrate. The similarity of the dose relation of these two compounds and their effectiveness in enhancing mortality caused by the infection is clearly seen. The correlation coefficient was highly significant (p -0.001) for mortality as well as the mean survival time (see Fig. 6).
The possible neutralizing effect of the ammonium ion observed in the zinc ammonium sulfate was also seen in preliminary experiments with aluminum sulfate and aluminum ammonium sulfate. As shown in Table 4, exposure to 2.5 mg/m3 of aluminum sulfate resulted in a significant increase in mortality and decrease in mean survival time. On the other hand, a similar 3-hr exposure to the same concentration of aluminum ammonium sulfate resulted in somewhat increased mortality which, however, was not statistically significant. Figure 7 shows the relationship between the concentration of several sulfates and excess mortality.
The least-square lines are based on linear regression analyses of excess mortalities resulting from a single 3-hr exposure to these compounds and infectious challenge. It is apparent from these data that cadmium sulfate was most effective and magnesium sulfate least effective in reducing the resistance to streptococcal pneumonia. The estimated concentrations of the compounds which induced 20% excess mortality (ED20) were 0.2 mg/m3 cadmium sulfate, 0.6 mg/m3 copper sulfate, 1.5 mg/m3 zinc sulfate, 2.2 mg/m3 aluminum sulfate, 2.5 mg/m3 zinc ammonium sulfate, and 3.6 mg/m3 magnesium sulfate. Estimated on the same basis, exposure to 1.3 mg/m3 of zinc nitrate similarly resulted in 20% excess mortality. Table 5 shows preliminary data on the maximum concentrations of sulfates and nitrates tested to-date which, after a 3-hr exposure, did not result in significant changes in mortality or survival time in mice challenged with Streptococcus aerosol.
The data shown in Figure 7 and Table 5 add another dimension to this animal model system. It is S04, m9/m 3 FIGURE 7. Excess mortality in mice exposed for 3 hr to sulfates and challenged with Streptococcus aerosol. Environmental Health Perspectives g6 apparent that changes in resistance to respiratory infection can be used to rank the effects of inhalation of pollutants having a related chemical structure. In all experiments the exposure conditions, including the particle size of the aerosol, were kept identical. Thus, the differences in response can only be ascribed to the toxicity of the compounds.

Experimental Variables
Several experimental variables can affect the responses in this animal model system. They include the animal host, infectious agent, and exposure sequences.

Animals
The selection of the animal host in this model system is of a major importance. Figure 8 shows the response of mice, hamsters and squirrel monkeys exposed to nitrogen dioxide and challenged with K. pneumoniae aerosol (5) as the percent mortality in the three species of animals and the numbers of animals used to obtain these means. Within each animal species, the mortality data were subdivided into three groups. The first is mortality of the control group of animals challenged with the infectious agent but exposed to filtered air. The second group represents mean mortality of animals challenged with the infectious agent and exposed to concentrations of nitrogen dioxide which did not significantly enhance the mortality. The third group is the mean mortality of animals at nitrogen dioxide concentrations at which significant enhancement in mortality was observed.
The mortality of control animals indicates the range of natural resistance to bacterial pneumonia in different species of animals. The respective death rates in control mice, hamsters, and monkeys were 41%, 1 1%, and 0o. The respiratory dose for monkeys and hamsters was approximately 105 organisms, a dose of the infectious agent which repeatedly killed the Swiss albino mice. Thus, the higher concentrations of nitrogen dioxide necessary to induce a significant increase in mortality in mon- keys and hamsters can, at least in part, be ascribed to their innate resistance to this infectious agent. Differences in natural resistance to bacterial pneumonia were also seen in studies of the effects of nitrogen dioxide in four strains of mice and are shown in Table 6. The BDF mice showed significantly higher natural resistance to the infection than the other strains. However, excess mortalities ranging from 8 to 25% were observed in all four strains of mice after 3-hr exposure to 5 ppm of N02 and the infectious challenge. Table 7 shows the effects of a single 2or 3-hr exposure to nitrogen dioxide on the resistance of Swiss albino mice to bacterial pneumonia induced by inhalation of K. pneumoniae or S. pyogenes aero- a. b, cMeans in a column with the same superscript are not significantly different (p s 0.05). dSignificant change in mortality compared to corresponding infected mice exposed to filtered air (p S 0.05).

Exposure Sequence
The sequence and interval between the exposure to the pollutants and challenge with the infectious agent is yet another factor of importance. In all of the studies discussed previously the experimental plan required exposure to the pollutants followed within less than I hr by the infectious challenge. Figure 9 shows that, at least for the short-term exposures to nitrogen dioxide the extension of the interval between exposure and challenge allows a recovery from the effects of the pollutants.

Conclusions
In discussing the importance of this animal model system in studies of health effects of air pollutants two aspects must be considered.
First is the value of the model as a highly sensitive indicator ofin vivo effects. The changes in resistance to respiratory infection can be seen at concentrations of oxidant and other pollutants markedly lower than those detected by any other in vivo method. The model depicts the responses of the respiratory system and reflects the overall damage to the pulmonary defense mechanisms. Therefore, it indicates that basic defense mechanisms of the lungs have been impaired. A major part of this impairment appears to result from the damage to the alveolar macrophage system and the ensuing inability to remove the inhaled microorganisms from the lungs.
The second aspect is that this model may simulate a situation which could occur in man. Decreased resistance to respiratory infections as a consequence of exposture to air pollutants was observed in mice, hamsters and monkeys. In spite of the functional and anatomical differences of the respiratory tract and the differences in innate and acquired immunity between man and these animal species, under the proper set of circumstances man could be expected to respond to these stresses in a very similar manner. The necessary components for its occurrence in man are the presence of a sufficiently high concentration of pollutants and the presence of an infectious microorganism capable to invade and colonize in the human host exploiting the state of reduced resistance. 6 27 FIGURE 9. Effect of time interval between 2 hr exposure to N02 and infectious challenge.
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