Absence of the macrophage mannose receptor in mice does not increase susceptibility to Pneumocystis carinii infection in vivo

Abstract

Host defense against the opportunistic pathogen Pneumocystis carinii requires functional interactions of many cell types. Alveolar macrophages are presumed to be a vital host cell in the clearance of P. carinii, and the mechanisms of this interaction have come under scrutiny. The macrophage mannose receptor is believed to play an important role as a receptor involved in the binding and phagocytosis of P. carinii. Although there is in vitro evidence for this interaction, the in vivo role of this receptor in P. carinii clearance in unclear. Using a mouse model in which the mannose receptor has been deleted, we found that the absence of this receptor is not sufficient to allow infection by P. carinii in otherwise immunocompetent mice. Furthermore, when mice were rendered susceptible to P. carinii by CD4(+) depletion, mannose receptor knockout mice (MR-KO) had pathogen loads equal to those of wild-type mice. However, the MR-KO mice exhibited a greater influx of phagocytes into the alveoli during infection. This was accompanied by increased pulmonary pathology in the MR-KO mice, as well as greater accumulation of glycoproteins in the alveoli (glycoproteins, including harmful hydrolytic enzymes, are normally cleared by the mannose receptor). We also found that the surface expression of the mannose receptor is not downregulated during P. carinii infection in wild-type mice. Our findings suggest that while the macrophage mannose receptor may be important in the recognition of P. carinii, in vivo, this mechanism may be redundant, and the absence of this receptor may be compensated for.

FIG. 1.

Inflammatory cells in the BAL of P. carinii-inoculated MR-KO mice (grey bar), wild type (WT) mice (slashed bar), and uninfected wild-type (CON, panel A only) (open bar) mice. (A) Ten days after i.t. inoculation; (B) 28 days after i.t. inoculation; (C) after 35 days of continuous exposure to P. carinii via cohabitation with previously infected SCID mice. Cell types are macrophages (MØ), neutrophils (PMN), CD4⁺ lymphocytes (CD4⁺), and CD8⁺ lymphocytes (CD8⁺). *, MR-KO mice are significantly different from WT mice (P ≤ 0.05). Values are means ± standard error of the mean; n = 4 to 6.

FIG. 2.

Pneumocystis levels in the lungs of MR-KO and wild-type mice that have been immunocompromised by the depletion of CD4⁺ lymphocytes. Mice were inoculated i.t. with 10⁷ P. carinii nuclei. At the indicated days, the pulmonary pathogen load was determined by visual counts of P. carinii nuclei from lung homogenate samples. □, MR-KO; ○, wild type; ▪, wild-type uninoculated control. Values are means ± standard deviation; n = 5 except for MR-KO 10-day (n = 6), and MR-KO 21-day (n = 4) results.

FIG. 3.

Inflammatory cells in the BAL of CD4⁺ lymphocyte-depleted and P. carinii-inoculated MR-KO (grey bar) mice, wild-type (WT) mice (slashed bar), and nondepleted, uninfected wild-type control (CON) mice (open bar). (A) Ten days after i.t. inoculation; (B) 21 days after inoculation; (C) 28 days after inoculation; (D) 35 days after inoculation. Cell types are macrophages (MØ), neutrophils (PMN), and CD8⁺ lymphocytes (CD8⁺). *, MR-KO mice are significantly different from WT mice (P ≤ 0.05). Values are means ± standard of the mean; n = 5.

FIG.4.

Respiratory pathology in CD4⁺ lymphocyte-depleted and P. carinii-infected MR-KO and wild-type (WT) mice. (A) Albumin concentration in 5-ml lavage samples. (B) Total protein concentration in lavage samples. (C) LDH concentration in 5-ml lavage samples. (D) Rate of respiration of mice prior to tissue sampling. (E) Partial pressure of O₂ in blood sampled from the tail artery immediately prior to sacrifice. □, MR-KO; ○, WT; ▪, wild-type uninoculated control. Values are means ± standard deviation; n = 4 to 6. x axis refers to number of days since inoculation with P. carinii. *, MR-KO mice are significantly different from WT mice (P ≤ 0.05).

FIG. 5.

Pulmonary histology in CD4⁺ lymphocyte-depleted and P. carinii-infected MR-KO and wild type (WT) mice. Shown are hematoxylin- and eosin-stained paraffin sections from formalin-fixed lungs of MR-KO (A), WT (B), and control untreated (C) mice. Samples are from mice 35 days post-infection. Both MR-KO (A) and WT (B) lungs exhibited widespread alveolar filling with P. carinii organisms and mixed inflammatory cell infiltrate. Black bar in panel C, 100 μM. The three pictures are of equal magnification.

FIG. 6.

Total glycoproteins in BAL from CD4⁺-depleted and P. carinii-infected MR-KO and wild-type mice. Proteins in BAL samples were resolved on a 4 to 20% SDS polyacrylamide gel, periodate oxidized, and stained with a fluorescent probe. Each lane is sample pooled from three mice in each group. Lanes 1, 2, and 3 are, respectively, control (uninoculated wild-type) mice, infected wild-type mice, and infected MR-KO mice at 28 days postinoculation. Lanes 4, 5, and 6 are control mice, infected wild-type mice, and infected MR-KO mice at 35 days postinoculation.

FIG. 7.

Binding of mannosylated BSA to alveolar macrophages. BAL macrophages were incubated with mannose-BSA-biotin in the presence of calcium and then washed and incubated with streptavidin-APC. Geometric mean fluorescence of the cells was determined by flow cytometry. Cells are from mice that were CD4⁺ lymphocyte depleted and P. carinii infected for 28 days: MR-KO is infected MR-KO mice, and WT is infected wild-type mice. Control cells are from wild-type mice that have not been depleted or infected. 2°Only refers to cells from wild-type-infected mice that were incubated with streptavidin-APC but not mannose BSA. Bars represent mean ± standard error of the mean; n = 5. *, MR-KO mice are significantly different from WT mice (P ≤ 0.01).