Flow cytometric analysis of immune cell populations in the bronchial and mesenteric lymph nodes of the dromedary camel

Abstract

Dromedary camel is an important livestock species with special economic value in arid and semi-arid regions of the world. Given the limited data on detailed immune cell composition and cell marker expression in the dromedary camel lymph node tissue, the present study was undertaken to investigate the immune cell composition of bronchial and mesenteric lymph nodes from healthy dromedary camels using flow cytometry. In this study, we applied flow cytometry and multicolor immuno-fluorescence to phenotype the main populations of immune cells in the bronchial and mesenteric camel lymph nodes and compared them with separated peripheral blood mononuclear cells and granulocytes. We used antibodies to detect several cell surface molecules associated with camel T cells (CD4, WC1), B cells (MHCII, BAQ44A), monocytes/macrophages (CD172a, CD14, CD163), in addition to the pan-leukocyte marker CD45 and the cell adhesion molecules CD44 and CD18. Compared to blood mononuclear cells, camel lymph node cells contained a higher percentage of lymphoid cells with only a minor fraction of myeloid cells. In addition, the lower expression of CD44 and CD18 on lymph node lymphocytes compared to lymphocytes from peripheral blood indicates higher frequency of naïve lymphocytes in the lymph nodes. The frequency of CD4⁺ T cells, B cells and γδ T cells within camel lymph node lymphocytes compared to blood indicates a similar tissue distribution pattern of lymphocyte subsets in camel and bovine and supports previous reports on the similarity between the camel immune system and the immune system of other ruminants. Lymph node neutrophils were identified as CD45⁺⁺ CD172a⁺⁺, CD14⁺, MHCII^low, BAQ44A⁺, CD44⁺⁺, CD18⁺⁺ cells. In conclusion, the present study is describing the employment of flow cytometric single-cell analysis and immunostaining for the analysis of the immune cell composition in the camel lymph node.

Keywords: B cells; T cells; camel; flow cytometry; immune system; lymph node.

Figure 1

Gating strategy for the flow cytometric analysis of blood and lymph node (bronchial and mesenteric) mononuclear cells. Cells were stained with mAbs to CD45, CD172a, and CD14 and analyzed on the flow cytometer. (A) The percentage of cells stained positive with CD45 mAb was identified within single cells after excluding the cell doublets. (B) Staining of lymph node cells and blood MNC with mAbs to CD172a within CD45-positive cells. (C) Staining pattern of lymph node cells and blood MNC with mAbs to CD14. The percentage of lymphocytes (D) and monocytes (E) of total bronchial (blue color), mesenteric (red color) lymph node, or blood MNC (black color) were calculated and presented. * indicates significant differences (n = 10 animals for lymph node samples and six animals for blood samples; p < 0.05).

Figure 2

Lymphocyte subsets in bronchial and mesenteric lymph node and blood of camels. (A) After gating on lymphocytes within MNC, CD4+ and WC1+ cells were identified based on their staining with corresponding monoclonal antibody compared to isotype control staining. (B) Camel B cells were identified as MHCII-positive cells within the lymphocyte population (CD14-negative MNC). The percentages of CD4+ (C), WC1+ (D), and B cells (E) within lymphocytes were calculated and presented comparatively for bronchial (blue color), mesenteric (red color) lymph node, or blood MNC (black color).

Figure 3

Expression of the B cell marker BAQ44A on bronchial and mesenteric lymph node and blood lymphocytes. Lymph node cells or blood MNC were labeled with antibodies to CD14, MHCII, and BAQ44A. (A) The fraction of BAQ44A+ cells within lymphocytes was identified as CD14-BAQ44A+ cells. (B) After gating on lymphocytes (CD14- MNC), a gate was placed on MHCII+ cells and the percentage of BAQ44A+ cells within MHCII+ cells was identified in a separate dot plot. (C) The BAQ44A+ cells within total lymphocytes or within B cells (MHCII+ lymphocytes) were calculated and presented for bronchial (blue color), mesenteric (red color) lymph node, or blood MNC (black color).

Figure 4

Expression of the cell adhesion molecules CD44 and CD18 on camel lymph node and blood lymphocytes. Camel lymph node cells and blood MNC were stained with antibodies to camel CD44 or CD18 and analyzed by flow cytometry. Histogram presentation of CD44+ (A) or CD18+ (B) lymphocytes within bronchial lymph node (blue line), mesenteric lymph node (red line), and blood cells (black line) in comparison to isotype control (green line). Mean fluorescence intensity (MFI) of CD44+ cells (C) or CD18+ cells (D) were calculated and presented in scattered plot graphs for bronchial (blue color), mesenteric (red color) lymph node, or blood MNC (black color).

Figure 5

Gating strategy for lymph node and blood neutrophils (PMN). (A) Camel leukocytes or lymph node cells were analyzed by flow cytometry. (B) Neutrophils were identified based on their forward and side scatter properties and their percentages within bronchial (blue color), mesenteric (red color) lymph node, or blood leukocytes (black color) were presented (C).

Figure 6

The immunophenotype of lymph node and blood neutrophils. (A) Lymph node neutrophils were stained with mAbs to different cell markers and analyzed on the Accuri flow cytometer. (B) Blood-derived neutrophils were stained with mAbs to BAQ44A and analyzed by flow cytometry.

Figure 7

The abundance of CD45 (A), CD172a (B), CD14 (C), MHCII (D), CD44 (E), CD18 (F), and BAQ44A (G) on neutrophils from camel bronchial (blue color), mesenteric (red color) lymph node, or blood neutrophils (black color). Molecule expression level was presented as mean fluorescence intensity (MFI) of the corresponding antibody staining.