Immune and airway effects of house dust mite aeroallergen exposures during postnatal development of the infant rhesus monkey.

BACKGROUND: The effect of chronic environmental aeroallergen exposure on the immune system and airways has not been experimentally defined in very young children. OBJECTIVE: The purpose of this study was to determine the immunophenotype of peripheral blood and airway leucocytes in the newborn rhesus macaque monkey, following recurrent aerosol exposure to house dust mite (HDM) (Dermatophagoides farinae). METHODS: A regimen of HDM aerosolization was initiated for 2 h per day, three times per week, starting when rhesus macaque monkeys were 1 week of age. All monkeys were inoculated with diptheria, tetanus, and acellular pertussis vaccine at 5 weeks of age to simulate human infant vaccination schedules. RESULTS: Following 8 weeks of HDM aeroallergen exposure, infant monkeys exhibited a significant reduction in the total peripheral blood lymphocyte numbers and a decreased frequency of peripheral blood CD4+ T lymphocytes with a CD45RA-'memory' immunophenotype. Lavage CD4+ T lymphocytes from HDM-exposed monkeys showed elevated expression of CD25, as well as an increase in CD45RA-/CD62L-/CD11ahigh immunophenotype. Eosinophils were more abundant within airways of HDM-exposed monkeys, accumulating maximally within the trachea. CONCLUSION: These data demonstrate the development of immunological responses following chronic inhalation of a common environmental allergen during postnatal maturation in the non-human primate.

Lung collagen cross-links in rats with experimentally induced pulmonary fibrosis.

Rats were intratracheally instilled with bleomycin or with silica (quartz) dust to induce lung fibrosis. Several weeks later, purified collagen chains (or collagen digests) were isolated from the lungs of these animals and from age-matched controls instilled intratracheally with saline solution, and the ratios of hydroxylysine to lysine and of the dysfunctional cross-links DHLNL to HLNL were quantified. Collagen from fibrotic lungs had significantly higher ratios of DHLNL:HLNL than did control lungs, 15.5 +/- 4.8 and 17.1 +/- 4.8 vs. 2.3 +/- 0.5 for the silica-instilled and the bleomycin-instilled animals, respectively. The hydroxylysine:lysine ratio was significantly increased for the alpha 1(I) chain, to a value 170% of that of lung collagen from control animals, and for several of its constituent CNBr peptides. Lung tissue was exhaustively digested with collagenase and specific cross-linked peptides were isolated and characterized. The cross-linked alpha 1(I) x alpha 1(I) peptide linked by the residues 87 x 16C, with a ratio of DHLNL:HLNL of 17:1, demonstrated that the increased hydroxylation of the dysfunctional cross-links in fibrotic lung collagen could be accounted for in part by increased hydroxylation of the lysine residue at position 16C of the C-terminal telopeptide of the collagen alpha 1(I) chain. It proved impossible to locate the corresponding N-terminal cross-linked fragment from alpha 1(I) x alpha 1(I) chains, 9N x 930, possibly due to further reactions of this material to form the material referred to as poly(CB6). Isolated poly (CB6) accounted for more than half of the total alpha 1(I)CB6 peptide expected in lung collagen, and had a hydroxylysine:lysine content 2.8 times greater in bleomycin-treated animals than in their age-matched controls. Evidence was also found for a cross-linked alpha 1(III) x alpha 1(I) peptide linking residue 87 from the alpha 1(III) chain with residue 16C from the alpha 1(I) chain; it also had an increased ratio of DHLNL:HLNL. We conclude that the increased hydroxylation of lysine observed in two different animal models of lung fibrosis occurs preferentially at the N- and C-terminal nonhelical extension peptides of the alpha 1(I) collagen chains, and that this apparent specificity of overhydroxylation of fibrotic collagen may have important structural and pathological consequences.

Tendon hypertrophy is associated with increased hydroxylation of nonhelical lysine residues at two specific cross-linking sites in type I collagen.

This study was designed to investigate whether the changes in lysine hydroxylation known to occur in hypertrophic tendon occur randomly or at specific lysine residues in the type I collagen molecule. Peptides corresponding to the two known major cross-linking sites of type I collagen (a lysine (or hydroxylysine) at position 9N cross-linked to a hydroxylysine at 930 and a lysine (or hydroxylysine) at position 16C cross-linked to a hydroxylysine at position 87) were prepared by collagenase digestion, size fractionation, and separation by high performance liquid chromatography from normal chicken tendon and from chicken tendon subjected to increased tensile load as a result of muscle hypertrophy. The ratio of the difunctional cross-links dihydroxylysinonorleucine to hydroxylysinonorleucine in normal tendon is 0.75:1; this ratio is increased to 6:1 in hypertrophic tendon. The dihydroxylysinonorleucine to hydroxylysinonorleucine ratio is increased to the same extent in samples of the purified cross-linked peptides derived from both the N-terminal and C-terminal lysine aldehyde residues. On the other hand, the relative hydroxylysine content of preparations of the pooled larger helical peptides obtained by cyanogen bromide digestion of normal and hypertrophic tendons was essentially identical. These results demonstrate that there is a specific increase in hydroxylation of only the N- and C-terminal non-helical lysine residues that participate in the formation of the reducible difunctional cross-links of type I collagen in hypertrophic tendon, while the extent of hydroxylation of lysine residues in the helical regions is not affected. The specific mechanism by which the enzyme lysyl hydroxylase acting on its substrate can distinguish between lysine residues destined to be in non-helical versus helical regions in a nascent collagenous peptide that has not yet attained its final secondary structure remains to be defined.

Silica increases cytosolic free calcium ion concentration of alveolar macrophages in vitro.

Rat alveolar macrophages were exposed to silica dust (quartz) suspended in culture medium (SiO2, dry particle size less than 5 microns in diameter) and fluctuation in their cytosolic free calcium content ([Ca2+]i) was detected in cell monolayers with a fluorescent calcium probe (Indo-1AM). Cytosolic free calcium content was correlated with lactate dehydrogenase (LDH) release, an index of cell damage. SiO2 induced a concentration- and time-dependent increase of cytosolic free Ca2+ ion concentration and LDH release. [Ca2+]i was increased about fivefold when cells were exposed to 200 micrograms of SiO2 per milliliter (3 ml per dish) for 2 hr. [Ca2+]i changed within 15 min of SiO2 treatment, whereas LDH release was measurably increased only after 30 min. Chelation of extracellular Ca2+ by 2 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate did not prevent SiO2-induced fluctuation of macrophage [Ca2+]i, but did partially prevent the SiO2-induced increase in LDH release (p less than 0.01). We conclude that a very early event in SiO2-induced damage of alveolar macrophages involves mobilization of intracellular calcium pools to increase [Ca2+]i. These results suggest that SiO2-induced macrophage damage, a key event in the development of silicosis, may involve perturbation of intracellular calcium homeostasis.

Hydroxylation of collagen by lungs of rats administered bleomycin.

Collagen synthesized by tissue minces from lungs of rats administered 1 unit of bleomycin by intratracheal instillation 1 or 2 wk earlier contained relatively more hydroxylysine than did collagen made by lungs from saline-instilled control animals. Most, if not all, of the relative increase in lysine hydroxylation could be localized to the alpha 1 (I) chain of type I collagen. Lung homogenates from bleomycin-treated rats showed increased activity of lysyl hydroxylase (EC 1.14.11.4), the enzyme catalyzing the conversion of collagen-bound lysine to hydroxylysine. Thus, the increased hydroxylation of lysine and of lysine-derived cross-links previously observed in collagen of diseased human lungs and in animal models of lung fibrosis is reflected in an in vitro system.

Lung collagen and elastin after ozone exposure in vitamin B-6-deficient rats.

The effects of vitamin B-6 deficiency and ozone exposure on selected features of connective tissue metabolism in lung were investigated in groups of weanling male rats fed one of three diets: B-6-supplemented, fed ad lib; B-6-deficient, fed ad lib; or B-6-supplemented, restricted to the food intake of deficient rats for 5 weeks. Also, perinatal rat pups were studied that were nursed from dams fed one of the 3 diets from parturition to day 15 of lactation. During the final week of each experiment, half of the rats in each of the groups were exposed to 0.64 ppm of ozone (23.5 h per day). The collagen and elastin content, collagen synthesis rate, total protein synthesis rate, and lysyloxidase activity of lungs were measured. Perinatal pups rendered vitamin B-6-deficient were particularly sensitive to ozone exposure (65% died as compared to fewer than 5% of the ad lib or food-restricted controls). When L-proline incorporation into collagen and total protein was investigated using lung minces, food restriction and B-6-deficiency resulted in about one-half the incorporation normally observed. Total lung lysyl oxidase activity was also decreased in B-6-deficient and food-restricted rats compared to B-6-supplemented rats fed ad lib. Exposure to ozone resulted in increased lysyl oxidase activity and collagen synthesis in lungs from B-6-supplemented rats, but such responses were not observed in B-6-deficient or food-restricted (FR) rats exposed to ozone.

Deferoxamine injection does not affect bleomycin-induced lung fibrosis in rats.

Bleomycin is an antineoplastic agent that causes a dose-related lung fibrosis that limits its therapeutic effectiveness. It has been proposed that the cellular toxicity and antitumor effects of bleomycin occur by formation of O2-Fe(II)-bleomycin complexes that degrade DNA and release O2- and OH radicals that attack other cellular components. Twice daily injections of the iron chelator deferoxamine were utilized in an attempt to ameliorate bleomycin-induced lung fibrosis. They failed to diminish bleomycin-induced lung inflammation and fibrosis in rats.

Ozone exposure, food restriction and protein deficiency: changes in collagen and elastin in rodent lung.

Two groups of weanling or young adult rats were fed ad lib casein-based diets containing 4 or 16% protein. Food was restricted in a third group (fed the 16% protein diet) to the amount consumed daily by rats (adult or weanlings) fed the 4% diet. After 3 weeks (weanlings) or 1, 3 or 5 weeks (adults), one-half of the rats in each group were exposed to 0.64 ppm (1.28 mg/m3) of ozone for 7 days (23.5 h each day). Several parameters were then evaluated related to lung connective tissue metabolism including: (1) total lung hydroxyproline, (2) total lung elastin, (3) apparent rates for lung collagen synthesis and elastin accumulation and (4) lung and body weights. In general, the response to protein deficiency and food restriction was more pronounced than to ozone exposure. Protein deficiency and food restriction resulted in decreased lung size and collagen content. However, the ability of lung to respond to ozone (in relative terms) was not altered by changes in diet as assessed by changes in lung weight or the collagen synthetic rate.

Synergistic effects on rat lungs of mixtures of oxidant air pollutants (ozone or nitrogen dioxide) and respirable aerosols.

A hitherto unexpected synergism between the oxidant air pollutants ozone or nitrogen dioxide and a respirable-sized aerosol of ammonium sulfate was observed during controlled exposures of rats to these substances. Response of rat lungs to these pollutants was quantitated by measurement of apparent collagen synthesis rates in vitro by lung minces from exposed animals. Dose-response curves to either O3 or NO2 were altered in the presence of 5 mg/m3 of (NH4)2SO4 aerosol. Morphometric and histologic observations of lungs from rats exposed to high levels of ozone, with and without concurrent exposure to the (NH4)2SO4 particles, confirmed such synergistic effects. In a separate set of experiments, rats were exposed at near ambient levels to mixtures of ozone and sulfuric acid aerosol (submicron-sized aerosol). Potentiation of ozone effects on lung collagen synthesis rates was also observed in these experiments. These observations may have broad implications for the appropriate evaluation of laboratory data in the setting of ambient air quality standards and/or threshold limit values for occupational safety.