SARS spike protein induces phenotypic conversion of human B cells to macrophage-like cells

Abstract

Massive aggregations of macrophages are frequently detected in afflicted lungs of patients with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In vitro, ectopic expression of transcription factors, in particular CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPβ, can convert B cells into functional macrophages. However, little is known about the specific ligands responsible for such phenotype conversion. Here, we investigated whether spike protein of SARS-CoV can act as a ligand to trigger the conversion of B cells to macrophages. We transduced SARS-CoV spike protein-displayed recombinant baculovirus (SSDRB), vAtEpGS688, into peripheral B cells and B lymphoma cells. Cell surface expression of CD19 or Mac-1 (CD11b) was determined by flow cytometry. SSDRB-mediated changes in gene expression profiles of B lymphoma cells were analyzed by microarray. In this report, we showed that spike protein of SARS virus could induce phenotypic conversion of human B cells, either from peripheral blood or B lymphoma cells, to macrophage-like cells that were steadily losing the B-cell marker CD19 and in turn expressing the macrophage-specific marker Mac-1. Furthermore, we found that SSDRB enhanced the expression of CD86, hypoxia-inducible factor-1α (HIF1α), suppressor of cytokine signaling (SOCS or STAT-induced STAT inhibitor)-3, C/EBPβ, insulin-like growth factor-binding protein 3 (IGFBP3), Krüpple-like factor (KLF)-5, and CD54, without marked influence on C/EBPα or PU.1 expression in transduced cells. Prolonged exposure to hypoxia could also induce macrophage-like conversion of B cells. These macrophage-like cells were defective in phagocytosis of red fluorescent beads. In conclusion, our results suggest that conversion of B cells to macrophage-like cells, similar to a pathophysiological response, could be mediated by a devastating viral ligand, in particular spike protein of SARS virus, or in combination with severe local hypoxia, which is a condition often observed in afflicted lungs of SARS patients.

Fig. 1

Transduction specificity of SSDRB. (A) SSDRB (vAtEpGS688), which carried SARS-CoV spike protein on viral envelope and enhanced green fluorescent protein (EGFP) gene in viral genome, transduced isolated murine ATII cells. The average transduction rate at 100 MOI, as measured by the presence of EGFP⁺ cells, was around 35%. (B) SSDRB (vAtEpGS688) transduced ATII-derived lung adenocarcinoma cells, H226. The average transduction rate at 100 MOI was 16%.

Fig. 2

SSDRB transduction of human peripheral B cells. (A) Peripheral white blood cells (WBCs) were transduced with SSDRB (50 MOI), and EGFP⁺ transduced cells were observed by fluorescence microscopy. (B) Human WBCs were transduced with SSDRB (50 MOI) and observed by fluorescence microscopy at 24, 48, and 120 h, respectively. (C) SSDRB (50 MOI) transduced anti-CD19-magnetic bead-enriched human peripheral B cells (right panel). Transduction of CD19⁻ WBCs was minimal (left panel). The appearance of EGFP⁺ B cells was larger and more granular than that of normal B cells at 24 h posttransduction. Cell morphology was similar to that of macrophages (center and upper right panels). (D) Human peripheral B (CD19⁺) cells were transduced with SSDRB (50 MOI), and analyzed by flow cytometry at 24 h posttransduction. Cells were gated for GFP fluorescence. Both transduced (GFP-positive) and untransduced (GFP negative) cells were analyzed for the expression of CD19 and Mac-1 (CD11b).

Fig. 2

SSDRB transduction of human peripheral B cells. (A) Peripheral white blood cells (WBCs) were transduced with SSDRB (50 MOI), and EGFP⁺ transduced cells were observed by fluorescence microscopy. (B) Human WBCs were transduced with SSDRB (50 MOI) and observed by fluorescence microscopy at 24, 48, and 120 h, respectively. (C) SSDRB (50 MOI) transduced anti-CD19-magnetic bead-enriched human peripheral B cells (right panel). Transduction of CD19⁻ WBCs was minimal (left panel). The appearance of EGFP⁺ B cells was larger and more granular than that of normal B cells at 24 h posttransduction. Cell morphology was similar to that of macrophages (center and upper right panels). (D) Human peripheral B (CD19⁺) cells were transduced with SSDRB (50 MOI), and analyzed by flow cytometry at 24 h posttransduction. Cells were gated for GFP fluorescence. Both transduced (GFP-positive) and untransduced (GFP negative) cells were analyzed for the expression of CD19 and Mac-1 (CD11b).

Fig. 3

SSDRB transduction-induced conversion of B lymphoma cells into macrophages. (A) As shown by fluorescence microscopy, SSDRB (50 MOI) transduced B lymphoma cells, CA46, HT, and Toledo. The appearance of EGFP⁺ cells was larger and more granular than that of the untransduced B lymphoma cells. (B) B lymphoma cells were transduced with SSDRB at 10 and 50 MOI, respectively. (C) SSDRB-treated CA46 and Toledo cells were analyzed by flow cytometry for the expression of Mac-1 and CD19 at 24 h posttransduction. SSDRB-treated cells were gated for GFP fluorescence (right panel). (D) Immunocytochemical characterization showed that transduced B lymphoma cells expressed the macrophage-specific marker CD68 from 24 to 48 h. These CD68-positive cells continuously increased in size from 24 to 48 h, and became morphologically like monocyte/macrophages with enlarged and horseshoe-shape nuclei (counterstained in blue with hematoxylin) (arrowhead). White arrow indicates an untransduced B lymphoma cell. Each experiment was repeated at least 3 times.

Fig. 3

SSDRB transduction-induced conversion of B lymphoma cells into macrophages. (A) As shown by fluorescence microscopy, SSDRB (50 MOI) transduced B lymphoma cells, CA46, HT, and Toledo. The appearance of EGFP⁺ cells was larger and more granular than that of the untransduced B lymphoma cells. (B) B lymphoma cells were transduced with SSDRB at 10 and 50 MOI, respectively. (C) SSDRB-treated CA46 and Toledo cells were analyzed by flow cytometry for the expression of Mac-1 and CD19 at 24 h posttransduction. SSDRB-treated cells were gated for GFP fluorescence (right panel). (D) Immunocytochemical characterization showed that transduced B lymphoma cells expressed the macrophage-specific marker CD68 from 24 to 48 h. These CD68-positive cells continuously increased in size from 24 to 48 h, and became morphologically like monocyte/macrophages with enlarged and horseshoe-shape nuclei (counterstained in blue with hematoxylin) (arrowhead). White arrow indicates an untransduced B lymphoma cell. Each experiment was repeated at least 3 times.

Fig. 4

SSDRB-mediated changes of gene expression profiles in B lymphoma cells. Microarray was used to identify genes that were expressed in Toledo cells after SSDRB (50 MOI) transduction. (A) Following analyses of scatter plotting and hierarchical clustering, differentially expressed genes were identified. Red indicates genes that were upregulated. Green indicates genes that were downregulated. Unchanged genes were shown in black. (B) Expression of CD86 and HIF1α increased markedly within 12 h of SSDRB treatment. At 48 h after SSDRB addition, the level of CD86 and HIF1α increased to more than 4-fold and 2.5-fold, respectively. Expression of SOCS-3, C/EBPβ, IGFBP3, KLF-5, and CD54 increased gradually around 24–48 h after SSDRB treatment. (C) Folds of gene enhancement (48 h vs. 0 h) were compared between SSDRB-treated cells and vAtE control. Each experiment was repeated at least 3 times.

Fig. 5

Exposure to hypoxia induces conversion of B lymphoma cells to macrophages. (A) Following exposure to hypoxia (in an incubation chamber with 5% CO₂ and 95% nitrogen) for 16–24 h, the size of some B lymphoma cells (as determined by Giemsa stain), both CA46 and Toledo, was increased markedly (indicated by arrows). Cell morphology of enlarged cells resembled that of monocytes/macrophages with horseshoe-shape nuclei. These cells were immunologically positive for CD68. (B) SSDRB-transduced B lymphoma cells (Toledo) (EGFP⁺ cells) could hardly engulf any red fluorescent beads (microspheres) under both normoxia and hypoxia exposures. In contrast, macrophages isolated from mouse peritoneal cavity could readily ingest red fluorescent microspheres with high efficiency. (C) Phagocytosis percentage (%) of SSDRB-transduced B lymphoma cells (Toledo) under normoxia or hypoxia was determined, respectively, as the percentage of cells positive with more than 2 internalized microspheres. ***Significantly different (P < 0.001). Each experiment was repeated at least 3 times.