Activated pulmonary macrophages are insufficient for resistance against Pneumocystis carinii

Abstract

CD4+ T cells are pivotal for elimination of Pneumocystis carinii from infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host of P. carinii organisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (A beta(-/-)), T-cell receptor alphabeta cells (TCR beta(-/-)), or all mature T and B lymphocytes (RAG-1(-/-)) were naturally susceptible to P. carinii, whereas mouse mutants lacking the gamma interferon (IFN-gamma) receptor (IFN-gamma-R(-/-)) or tumor necrosis factor alpha (TNF-alpha) type I receptor (p55) (TNF-alpha-RI(-/-)) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-gamma, TNF-alpha, and IL-12 in diseased TCR beta(-/-) and RAG-1(-/-) mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid A beta(-/-) mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-gamma nor TNF-alpha appeared to be essential for resisting natural infection with P. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4+ T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4+ TCR alphabeta cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4+ T lymphocytes, must contribute to successful resistance against P. carinii.

FIG. 1

P. carinii detection by histology and PCR. P. carinii organisms were undetectable by silver methenamine staining of lung sections from parasite-exposed healthy mutants (A, represented by IFN-γ-R^−/−), in contrast to morbid mutant mice (B, represented by TCRβ^−/−). Magnification of the diseased lung section is shown to distinguish stained sporangia (C). Whole lung digests were used for detection of P. carinii by PCR, products of which were blotted on nitrocellulose and hybridized with a P. carinii-specific gene fragment (D). Parasitized lung tissues of TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutant mice gave positive signals, whereas tissues from P. carinii-exposed heterozygous control mice (+/−) and from exposed IFN-γ-R^−/− or TNF-α-RI^−/− mutants did not reveal a detectable PCR signal. Bars, 50 μm.

FIG. 2

Expression of SR and MR in lung tissues and BAL cells of diseased mouse mutants. Cells in lung sections of diseased mutants (A, represented by TCRβ^−/−) exhibited profound SR expression, as determined by immunohistology, whereas none were detected in lungs of healthy animals (B, represented by TCRβ^−/−). Histochemical staining of cytospins of BAL cells from diseased mice identified expression of SR to be restricted to alveolar macrophages (C, represented by TCRβ^−/−). Labeling cytospins of BAL cells with FITC-conjugated mannopyranosyl-phenyl isothiocyanate from diseased (D, represented by TCRβ^−/−) and healthy (E, represented by TCRβ^−/−) mice illustrates constitutive MR expression by macrophages and granulocytes. (F) Unstained cells to control for green autofluorescence. Panels A to C represent AP-conjugated anti-SR type I/II MAb developed with NBT-BCIP and counterstained with nuclear fast red. Bars, 50 μm.

FIG. 3

Detection of iNOS expression in lung sections and cytospins of BAL cells by immunohistology. Lung sections of diseased TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutant mice consistently stained positively with a polyclonal Ab against iNOS (A, represented by Aβ^−/−). Lung sections from P. carinii-exposed IFN-γ-R^−/− (B) and TNF-α-RI^−/− (C) mutant mice were iNOS negative. Staining of cytospin preparations of BAL cells derived from parasitized mutant mice revealed that iNOS expression was restricted to alveolar macrophages (D, represented by Aβ^−/−). Arrow, multinucleated giant cell. (A to C) AP-conjugated anti-iNOS polyclonal Ab developed with NBT-BCIP and counterstained with nuclear fast red; (D) AP-conjugated anti-iNOS Ab developed with NBT-BCIP-INT and counterstained with hematoxylin. Bars, 50 μm.

FIG. 4

NO production by BAL cells in the presence or absence of SOD. BAL cells from diseased TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutants produced high levels of NO, whereas cells from nonparasitized mice did not (A). NO production by pulmonary cell cultures with (black bars) and without (white bars) SOD were determined (B). Corresponding cultures with and without SOD were performed with cells from the same mice. SOD eliminates secreted SO, therefore preventing SO from scavenging NO. Differences of NO production with and without SOD of each mutant strain are therefore used as an indication of SO secretion by the BAL cells. Shown are means of duplicates of three mice per mutant strain; error bars represent standard deviations. Results refer to 10⁵ pulmonary cells. h, healthy; d, diseased.

FIG. 5

Respiratory burst of BAL cells from healthy and P. carinii-parasitized mutant mice. (A) Respiratory burst by BAL cells from healthy mice stimulated with zymosan A. Cells from mouse strains without zymosan A did not show any activity. Results are representative of duplicates using 5 × 10⁴ BAL cells. (B) Respiratory burst by BAL cells from diseased TCRβ^−/− and Aβ^−/− mutants with and without zymosan A. Results are representative of duplicates using 10⁵ BAL cells. RLU, relative light units.

FIG. 6

Constitutive IFN-γ (A), TNF-α (B), and IL-12 (C) production by 10⁵ BAL cells from healthy and diseased mouse mutants with indicated deficiencies. Each data point corresponds to one mouse, for which at least two replicates were performed. Average values are indicated by horizontal bars. Asterisks indicate significant differences (Student t test, P < 0.05) between cytokine levels attained by healthy and diseased mice of each mutant strain. Statistics are not applicable in panel A, since healthy animals do not produce any IFN-γ. h, healthy; d, diseased.

FIG. 7

IL-1 mRNA of BAL cells from healthy and diseased mutants. IL-1β and IL-1α mRNAs were expressed by BAL cells from diseased TCRβ^−/− (T), Aβ^−/− (A), and RAG-1^−/− (R) mutants but not by their healthy counterparts. β-Actin controls verify abundant total RNA content in all probes used. h, healthy; d, diseased. m, size markers.

FIG. 8

NK1.1 cells in BAL fluids of diseased mutant mice. BAL cells from diseased TCRβ^−/− (A), Aβ^−/− (B), and RAG-1^−/− (C) mutants were stained with FITC-conjugated anti-NK1.1 and phycoerythrin-conjugated anti-CD3 MAb and analyzed by fluorocytometry. Illustrated are cells within the lymphocyte gate. NK1.1 single-positive cells comprise 0.6 to 2% of total BAL cells for each mutant strain.