Scavenger receptor A dampens induction of inflammation in response to the fungal pathogen Pneumocystis carinii

Abstract

Alveolar macrophages are the effector cells largely responsible for clearance of Pneumocystis carinii from the lungs. Binding of organisms to beta-glucan and mannose receptors has been shown to stimulate phagocytosis of the organisms. To further define the mechanisms used by alveolar macrophages for clearance of P. carinii, mice deficient in the expression of scavenger receptor A (SRA) were infected with P. carinii, and clearance of organisms was monitored over time. SRA-deficient (SRAKO) mice consistently cleared P. carinii faster than did wild-type control mice. Expedited clearance corresponded to elevated numbers of activated CD4(+) T cells in the alveolar spaces of SRAKO mice compared to wild-type mice. Alveolar macrophages from SRAKO mice had increased expression of CD11b on their surfaces, consistent with an activated phenotype. However, they were not more phagocytic than macrophages expressing SRA, as measured by an in vivo phagocytosis assay. SRAKO alveolar macrophages produced significantly more tumor necrosis factor alpha (TNF-alpha) than wild-type macrophages when stimulated with lipopolysaccharide in vitro but less TNF-alpha in response to P. carinii in vitro. However, upon in vivo stimulation, SRAKO mice produced significantly more TNF-alpha, interleukin 12 (IL-12), and IL-18 in response to P. carinii infection than did wild-type mice. Together, these data indicate that SRA controls inflammatory cytokines produced by alveolar macrophages in the context of P. carinii infection.

FIG. 1.

Clearance kinetics of Pneumocystis infection are relatively unchanged in SRAKO mice. Adult (A and B) or neonatal (C) wild-type and SRAKO mice were given lung inoculations of P. carinii, and lung burden was determined at various times postinfection. (A) C57BL/6 background; (B and C) 129/SvJ background. Data are means ± standard deviations for three to five mice per group. The numbers above the bars are numbers of mice per group that had a P. carinii burden below the limit of detection (log₁₀ 3.23). **, P < 0.001 for wild-type mice compared to SRAKO mice; *, P < 0.05 compared to wild-type controls at the same time point.

FIG. 2.

Elevated numbers of activated CD4⁺ T cells migrate into the lungs of adult or neonatal SRAKO mice in response to Pneumocystis infection. CD4⁺ cells with an activated phenotype (CD44^hi CD62L^lo) were enumerated from the BALF (A and D), lung digests (B and E), or draining lymph nodes (C and F) from mice infected with P. carinii as adults (A to C) or neonates (D to F) using flow cytometry. Data are means ± standard deviations for three to five mice per group. *, P < 0.05 compared to wild-type controls at the same time point.

FIG. 3.

Alveolar macrophages from SRAKO mice express elevated levels of CD11b. Mice were infected with P. carinii, and BALF (A and C) and lung digests (B and D) were collected at the indicated times. Flow cytometry was used to compare CD11b expression on CD11c⁺ macrophages. Dot plots from individual mice are representative of four mice per group at day 7 postinfection. The proportions of nonlymphocytes within gates R2, R3, and R4 are indicated. Data in panels C and D were gated on CD11c⁺ cells and include cells in gates R2 + R3. Data are means ± standard deviations of the MFI of cells from four mice per group. *, P < 0.05 compared to wild-type controls at the same time point.

FIG. 4.

The functional capacity of alveolar macrophages from SRAKO mice differs from wild-type macrophages. To quantitate the phagocytic capacity of alveolar macrophages, P. carinii organisms were labeled with DiO and inoculated into the lungs of wild-type or SRAKO mice. At 24 h, BALF cells were obtained and the proportion of DiO-associated cells determined by flow cytometry. The percent DiO⁺ cells (A) and MFI of DiO on individual macrophages (B) were not statistically different between wild-type and SRAKO cells.

FIG. 5.

Alveolar macrophage production of proinflammatory cytokines in response to P. carinii is decreased in SRAKO cells compared to wild type. To assess cytokine production by alveolar macrophages, cells were isolated from the BALF of uninfected mice and cultured for 24 h with LPS, sonicated P. carinii antigens (PC Ag), P. carinii organisms (PC), medium alone (naïve) (A and B), or zymosan (C). TNF-α (A and C) and IL-6 (B and C) were quantitated from culture supernatants with a cytokine bead array. Data are representative of replicate wells and three separate experiments.

FIG. 6.

P. carinii infection in SRAKO mice results in elevated proinflammatory cytokines in the lungs. Mice were infected with P. carinii, and BALF was analyzed for TNF-α (A) and IL-18 (B) by enzyme-linked immunosorbent assay. Data are means plus standard deviations for three to five mice per group and are representative of at least two separate experiments. *, P < 0.05 compared to wild-type controls at the same time point.