Severe acute respiratory syndrome (SARS) coronavirus-induced lung epithelial cytokines exacerbate SARS pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells

Abstract

Severe acute respiratory syndrome (SARS), which is caused by a novel coronavirus (CoV), is a highly communicable disease with the lungs as the major pathological target. Although SARS likely stems from overexuberant host inflammatory responses, the exact mechanism leading to the detrimental outcome in patients remains unknown. Pulmonary macrophages (Mphi), airway epithelium, and dendritic cells (DC) are key cellular elements of the host innate defenses against respiratory infections. While pulmonary Mphi are situated at the luminal epithelial surface, DC reside abundantly underneath the epithelium. Such strategic locations of these cells within the airways make it relevant to investigate their likely impact on SARS pathogenesis subsequent to their interaction with infected lung epithelial cells. To study this, we established highly polarized human lung epithelial Calu-3 cells by using the Transwell culture system. Here we report that supernatants harvested from the apical and basolateral domains of infected Calu-3 cells are potent in modulating the intrinsic functions of Mphi and DC, respectively. They prompted the production of cytokines by both Mphi and DC and selectively induced CD40 and CD86 expression only on DC. However, they compromised the abilities of the DC and Mphi in priming naïve T cells and phagocytosis, respectively. We also identified interleukin-6 (IL-6) and IL-8 as key SARS-CoV-induced epithelial cytokines capable of inhibiting the T-cell-priming ability of DC. Taken together, our results provide insights into the molecular and cellular bases of the host antiviral innate immunity within the lungs that eventually lead to an exacerbated inflammatory cascades and severe tissue damage in SARS patients.

FIG. 1.

SARS-CoV induces a dose- and time-dependent production of IL-6 and IL-8 by infected human bronchial Calu-3 cells. (A) Confluent Calu-3 cells grown in six-well plates with M-20 medium were infected with live SARS-CoV at MOIs of 5, 1, and 0.1 or, as a control, remained uninfected. The infected cultures were maintained in the M-20 medium before cell-free supernatants were harvested at day 4 after infection for assessing the IL-6 and IL-8 contents by ELISA. (B) Confluent Calu-3 cells were infected with live SARS-CoV (□) or gamma-inactivated (2 × 10⁶ rads) SARS-CoV (▵) at an MOI of 0.1 or remained uninfected (○) as control. The infected cultures were maintained with the M-20 medium and harvested at days 1, 2, 3, and 4 after infection, and their IL-6 content was assessed by ELISA. (C) Confluent CaLu-3 cells were infected with SARS-CoV (MOI = 0.1) for the indicated times before cellular lysates were extracted for immunoblotting for SARS-CoV nucleocapsid (NC) protein, IκB, and actin (as control), as described in Materials and Methods. Results are representative of at least three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01 (Student's t test) compared to different MOIs (A) or gamma-inactivated virus-infected controls (B). Error bars indicate SDs.

FIG. 2.

Apical supernatants derived from SARS-CoV-infected Calu-3 cells are potent in promoting cytokine production by Mφ. MO-derived Mφ were incubated for 3 days with 50% of gamma-inactivated apical supernatants harvested from either mock- or SARS-CoV-infected Calu-3 cells grown on the membrane inserts. The resulting cell-free supernatants were subjected to cytokine profiling by using the Bio-Plex Cytometric Bead Array assay. Data presented are the net values for the indicated cytokines produced by cultured Mφ, which were calculated by subtracting the baseline values existing in the apical supernatants harvested from those of infected Calu-3 cells. Data presented are means ± SDs. Results are representative of three independent experiments using Mφ derived from different individuals. *, P ≤ 0.05 (Student's t test) compared to those cultured with mock-infected supernatants.

FIG. 3.

Basolateral supernatants harvested from SARS-CoV-infected Calu-3 cells are potent in stimulating cytokine production by DC. MO-derived DC were cultured with 50% of gamma-inactivated supernatants harvested from the basolateral chambers of either mock- or SARS-CoV-infected Calu-3 cells, as described for Fig. 2, for a total of 3 days. The resulting supernatants were subjected to cytokine profiling by Bio-Plex analysis. Data presented are the net values for the indicated cytokines produced by cultured DC, which were calculated by subtracting the baseline values of the cytokines from those existing in the basolateral supernatants of infected Calu-3 cells. Results are representative of three independent experiments using DC derived from different individuals.

FIG. 4.

SARS-CoV-induced epithelial Calu-3 cell cytokines enhance the expression of CD40 and CD86 molecules on the surface of cultured DC. MO-derived DC were subjected to incubation with 50% of basolateral supernatants derived from either mock- or SARS-CoV-infected Calu-3 cells for 3 days. DC cultured in RPMI 1640-10% FCS medium were included as an additional control. The expression of costimulatory molecules was evaluated by fluorescence-activated cell sorter analysis. Data presented are the histogram plots from fluorescence-activated cell sorter analysis of cultured DC, showing the enhanced expression of CD40 and CD86. Results presented are representative of four independent experiments using DC derived from different individuals.

FIG. 5.

Apical supernatant harvested from SARS-CoV-infected Calu-3 cells reduces the phagocytic activity of Mφ. Mφ cultured with the apical supernatants derived from mock- or SARS-CoV-infected Calu-3 cells, or RPMI 1640-10% FCS medium, for 3 days, as described for Fig. 2, were harvested, and their capacity to take up FITC-conjugated dextran sulfate was analyzed by fluorescence-activated cell sorter analysis as described in Materials and Methods. Results are representative of three independent experiments using Mφ derived from different individuals.

FIG. 6.

The intrinsic T-cell-priming ability of DC is significantly suppressed subsequent to their cultivation with the basolateral supernatant derived from SARS-CoV-infected, but not mock-infected, Calu-3 cells. Aliquots of MO-derived DC were subjected to activation with LPS (5 μg/ml) or live SARS-CoV (MOI = 1) or cultivation with 50% of the basolateral supernatants harvested from mock- or SARS-CoV-infected Calu-3 cells for a total of 3 days. These differentially treated DC were evaluated for their capacity to prime naïve CD4⁺ T cells in the standard one-way MLR assay, as described in Materials and Methods. The proliferation of allogeneic T cells was expressed as the average cpm ± SD for triplicate samples. Data shown are representative of three independent experiments using DC and naïve T cells derived from different individuals *, P < 0.05 from Student's t test, in which we compared culture with supernatants derived from infected versus mock-infected Calu-3 cells.

FIG. 7.

IL-6 and IL-8 are crucial SARS-CoV-induced Calu-3 cell cytokines responsible, in part, for inhibiting the ability of DC to prime naïve T cells. (A) Aliquots of DC were incubated with M-10 medium alone or 50% of SARS-CoV-induced Calu-3 cell cytokines in the presence of control Ab and specific neutralizing Abs against IL-6, IL-8, or IP-10. (B) Additionally, they were incubated with appropriate concentrations of recombinant IL-6, recombinant IL-8, or recombinant IP-10. After cultivation for 3 days, DC were harvested to assess their ability to stimulate proliferation of naïve T cells in a standard MLR. The capacity of differentially treated DC is presented as the percentage of that elicited by M-10 medium-cultured DC. One-way analysis of variance with Bonferroni's multiple-comparison test was used to determine the level of statistical significance. Data presented are representative of at least two independent experiments. Error bars indicate SDs.