Opsonophagocytosis of Chlamydia pneumoniae by Human Monocytes and Neutrophils

Abstract

The human respiratory tract pathogen Chlamydia pneumoniae, which causes mild to severe infections, has been associated with the development of chronic inflammatory diseases. To understand the biology of C. pneumoniae infections, several studies have investigated the interaction between C. pneumoniae and professional phagocytes. However, these studies have been conducted under nonopsonizing conditions, making the role of opsonization in C. pneumoniae infections elusive. Thus, we analyzed complement and antibody opsonization of C. pneumoniae and evaluated how opsonization affects chlamydial infectivity and phagocytosis in human monocytes and neutrophils. We demonstrated that IgG antibodies and activation products of complement C3 and C4 are deposited on the surface of C. pneumoniae elementary bodies when incubated in human serum. Complement activation limits C. pneumoniae infectivity in vitro and has the potential to induce bacterial lysis by the formation of the membrane attack complex. Coculture of C. pneumoniae and freshly isolated human leukocytes showed that complement opsonization is superior to IgG opsonization for efficient opsonophagocytosis of C. pneumoniae in monocytes and neutrophils. Neutrophil-mediated phagocytosis of C. pneumoniae was crucially dependent on opsonization, while monocytes retained minor phagocytic potential under nonopsonizing conditions. Complement opsonization significantly enhanced the intracellular neutralization of C. pneumoniae in peripheral blood mononuclear cells and neutrophils and almost abrogated the infectious potential of C. pneumoniae In conclusion, we demonstrated that complements limit C. pneumoniae infection in vitro by interfering with C. pneumoniae entry into permissive cells by direct complement-induced lysis and by tagging bacteria for efficient phagocytosis in both monocytes and neutrophils.

Keywords: Chlamydia pneumoniae; complement; monocytes; neutrophils; opsonization; phagocytosis.

FIG 1

Complement opsonization of C. pneumoniae in nonimmune serum. Purified C. pneumoniae EBs were incubated in nonimmune serum (NHS−) (A) or heat-inactivated nonimmune serum (HIHS−) (B) and analyzed by immunoelectron microscopy (IEM) using anti-C3c antibodies and anti-rabbit antibody with 10-nm colloidal gold as primary and secondary antibodies, respectively. (C) Gold deposition on each bacterium was quantified relative to the background gold level for each condition (NHS− and HIHS−). (D) Purified C. pneumoniae EBs were subjected to Western blot analysis and labeled against C3c. C. pneumoniae EBs were incubated in NHS− (E) or HIHS− (F) and subjected to IEM using anti-C4c antibody as the primary antibody. (G) Complement C4 deposition was quantified as described for panel C. (H) Western blot analysis of complement C4 deposition on C. pneumoniae incubated in NHS− or HIHS−. Western blot analysis was repeated three times, and representative blots are shown. IEM images were captured from at least 8 random fields for each sample in one experiment. For each bacterium, a bacterium-to-background gold deposition ratio was calculated and plotted, with each dot representing one chlamydial organism. The median ratio is presented as black lines. Bars, 200 nm.

FIG 2

Antibody and complement opsonization of C. pneumoniae in immune serum. C. pneumoniae (5 × 10⁵ IFU/well) was grown in in HeLa cells for 48 h, and chlamydial inclusions were immunofluorescently stained using 2-fold serial dilutions of immune serum (A) or nonimmune serum (B) as the primary antibody. Purified C. pneumoniae EBs incubated in heat-inactivated immune serum (HIHS+) (C) or heat-inactivated nonimmune serum (HIHS−) (D) were immunolabeled with anti-human IgG with 10-nm colloidal gold and subjected to immunoelectron microscopy (IEM). (E) From IEM images, chlamydial gold deposition, for each bacterium, was quantified relative to the background gold level. Purified C. pneumoniae EBs were incubated in immune serum (NHS+) (F) or heat-inactivated immune serum (HIHS+) (G) and analyzed by IEM using anti-C3c antibodies and anti-rabbit antibodies with 10-nm colloidal gold as primary and secondary antibodies, respectively. (H) Gold deposition in IEM images was quantified as described for panel E. (I) Purified C. pneumoniae EBs were subjected to Western blot analysis and labeled against C3c. (J to M) C. pneumoniae was treated as described for panels F to I except the bacteria were labeled with an anti-C4c antibody. For immunofluorescence microcopy, each serum was tested at four different dilutions, and images were captured from five random fields for each dilution. The experiment was repeated twice. In IEM, a bacterium-to-background gold deposition ratio was calculated for each bacterium and plotted, with each dot representing one chlamydial organism. The median ratio is presented as black lines. IEM images were captured from at least 8 random fields for each sample in one experiment. Western blot analysis was repeated three times, and representative blots are shown. Bars, 10 μm (A and B) and 200 nm (C, D, F, G, J, and K).

FIG 3

Formation of membrane attack complex (MAC) on C. pneumoniae. Purified C. pneumoniae EBs were incubated in immune and nonimmune sera and processed for IEM. (A) C. pneumoniae incubated in immune serum (NHS+) and negatively stained with PTA. (B) C. pneumoniae incubated in immune serum (NHS+) and negatively stained with PTA. (C) C. pneumoniae incubated in nonimmune serum (NHS−) and immunogold labeled against C5b-9. Area with disintegrated bacterial morphology was enlarged (hatched boxes) demonstrating 10-nm pore-like structures (yellow arrowheads) (B and C) in close proximity to gold particles (C). (D) C. pneumoniae incubated in heat-inactivated immune serum (HIHS+) and immunogold labeled against C5b-9. Anti-mouse antibody with 10-nm colloidal gold was used as a secondary antibody. IEM images were used to quantify gold deposition on C. pneumoniae incubated in nonimmune serum (E) and immune serum (F). For each chlamydial organism, the gold particle deposition was quantified by calculating a bacterium-to-background ratio and the ratios were used to create scatterplots. Each dot represents the ratio from one chlamydial organism, and the black lines show the median ratios. Bars, 200 nm. HIHS−, heat-inactivated nonimmune serum.

FIG 4

Serum neutralization of C. pneumoniae EBs. C. pneumoniae inocula were incubated in immune serum (NHS+), nonimmune serum (NHS−), heat-inactivated immune serum (HIHS+), heat-inactivated nonimmune serum (HIHS−), or HIHS− supplemented with 1.5 mg/ml IgG from immune serum (HIHS−+IgG) for 30 min and used to infect HeLa cells. HeLa cells were inoculated with 10.8 × 10⁴ inclusion-forming units (IFU) of NHS-opsonized C. pneumoniae and 1.2 × 10⁴ IFU of HIHS-opsonized C. pneumoniae. IFU were quantified by immunofluorescence staining of chlamydial inclusions. Images were captured from seven random fields in each sample using a 16× lens objective. Data were obtained from duplicate samples from three independent experiments. All data are presented as means ± SDs. Differences between means were analyzed by Welch’s ANOVA with Games-Howell multiple-comparison test. *, P < 0.05.

FIG 5

Opsonophagocytosis of C. pneumoniae in monocytes and neutrophils. The percentages of C. pneumoniae-positive monocytes (A) and neutrophils (B) incubated with C. pneumoniae at an MOI of 10 in nonimmune serum were quantified by flow cytometry. The combined roles of complement and anti-C. pneumoniae IgG in monocyte (C) and neutrophil (D) phagocytosis were tested using immune serum. Heat-inactivated nonimmune serum and heat-inactivated nonimmune serum supplemented with the IgG fraction from immune serum were used as the controls to determine the role of IgG in phagocytosis in monocytes (E) and neutrophils (F). The percentages of C. pneumoniae-positive monocytes (G) and neutrophils (H) incubated with C. pneumoniae at an MOI of 1 in immune serum and nonimmune serum. Data were obtained from duplicate samples from three independent experiments except for those in panels E and F, which were obtained from duplicate samples from two independent experiments. A minimum of 8,000 gated events were obtained from each sample. All data are presented as means ± SDs. One-way ANOVA with Tukey’s post hoc test, Welch’s ANOVA with Games-Howell multiple-comparison test, or Welch’s t test was used to compare column means. *, P < 0.05; NHS+, immune serum; NHS−, nonimmune serum; HIHS+, heat-inactivated immune serum; HIHS−, heat-inactivated nonimmune serum; HIHS−+IgG, heat-inactivated nonimmune serum supplemented with IgG fraction from immune serum.

FIG 6

Intracellular neutralization of opsonized C. pneumoniae in PBMCs and neutrophils. C. pneumoniae (MOI = 10) was incubated with PBMCs (A) and neutrophils (B) under different culture conditions for 30 min, and cell lysates were prepared by ultrasonication. Lysates were used to infect HeLa cell monolayers, and inclusion-forming units (IFU) were enumerated after 48 h by immunofluorescence microscopy. Intracellular localization of C. pneumoniae in monocytes after 30 min was investigated by immunofluorescence staining and confocal microscopy of C. pneumoniae (C) and LAMP1 (D). (E) Overlay image. (F) Monocyte incubated with mock control. Confocal microscopy was repeated twice for all four (NHS+, NHS−, HIHS+, and HIHS−) conditions, and images were captured from five random fields in each sample. Bars, 5 μm. Quantitative data were obtained from duplicate samples from three independent experiments. Data are presented as means ± SDs. Differences between means were analyzed by Welch’s ANOVA with Games-Howell multiple-comparison test. *, P < 0.05; NHS+, immune serum, NHS−, nonimmune serum; HIHS+, heat-inactivated immune serum; HIHS−, heat-inactivated nonimmune serum.