Dynamics of M1 macrophages in oral mucosal lesions during the development of acute graft-versus-host disease in rats

Abstract

The role of macrophage infiltrates in oral mucosal acute graft-versus-host disease (AGVHD) remains unclear, although clinical studies suggest that macrophage infiltration correlates directly with the severity of AGVHD. In this study, we investigated the role of M1 macrophage infiltration in the oral mucosa of rats with AGVHD. Lewis rat spleen cells were injected into (Lewis × Brown Norway) F₁ rats to induce systemic GVHD. Tongue samples were evaluated using histology, immunohistochemistry, dual immunofluorescence, real-time reverse transcription-polymerase chain reaction, Transwell migration assays and Stamper-Woodruff binding assays. At the onset of oral mucosal AGVHD, dual immunofluorescence and migration assays revealed that M1 macrophages had accumulated in the basement membrane (BM) region via the laminin/CD29 β1 integrin pathway. Macrophage-secreted matrix metalloproteinase-2 was related to BM degradation. The adhesion of macrophages to the oral epithelium could be inhibited by pretreating macrophages with a CC chemokine receptor 2 (CCR2) antibody and/or pretreating lesion sections with monocyte chemoattractant protein-1 (MCP-1) antibody. Our data show that the migration and adhesion of M1 macrophages are associated with oral mucosal AGVHD, which is mediated in part by both laminin/CD29 β 1 intern and MCP-1/CCR2 pathways. Therefore, our study provides additional support for the contribution of macrophage infiltrate to the development of oral mucosal AGVHD.

Keywords: CC chemokine receptor 2; CD29 β 1 integrin; M1 macrophages; acute graft-versus-host disease; monocyte chemoattractant protein-1.

Figure 1

Assessment of oral mucosal acute graft‐versus‐host disease (AGVHD). (a) Weight curve in rats developing AGVHD. Rats were divided into three groups; control, phosphate‐buffered saline (PBS)‐treated rats; syngeneic, matched donor transplant, no AGVHD rats; and semi‐allogeneic, unmatched donor transplant, AGVHD rats. Weights are represented as the percentage of the mean ± standard deviation (s.d.) for each day compared with baseline for that group (weight on day 0). *Significantly different at P < 0·05 compared with control and syngeneic groups [analysis of variance (anova) followed by Scheffé's test]. Animal experiments performed in triplicate. (b) Histopathological and immunohistochemical assessments of oral mucosal AGVHD. Sections of both AGVHD and control tongues were stained with haematoxylin and eosin (H&E) and anti‐CD8 and anti‐ intercellular adhesion molecule‐1 (ICAM‐1) antibodies. Reactive products for immunostaining were developed using 5‐bromo‐4‐chloro‐3‐indolyl phosphate/nitro blue tetrazolium chloride solution (BCIP/NBT) solution. Arrows, mononuclear cells; arrow heads, epithelial vacuolation. Original magnification, ×200. Histological experiments performed in quadruplicate.

Figure 2

Activated macrophages accumulate and persist in oral mucosal acute graft‐versus‐host disease (AGVHD). (a) Immunofluorescent images of ED1‐positive macrophages in the tongue from the control and AGVHD‐mediated rats. Original magnification, ×200. For the quantitative analysis of macrophage numbers, 10 areas 50 µm² each in the tongue were selected randomly from each of the five rats and the total number of ED1‐positive cells was counted. The mean number of ED1‐positive cells per 10 areas ± standard deviation (s.d.) is shown for each group. *Significant difference at P < 0·01 (Student's t‐test). (b) Dual immunofluorescent images of ED1 (green) and inducible nitric oxide synthase (iNOS) (red) in oral mucosal AGVHD. The nucleus was stained with Hoechst 33324 (blue). Original magnification, ×200. (c) Distribution of M1 and M2 macrophages using iNOS (red) and ED2 (green) antibodies, respectively. Original magnification, ×200. Infiltration of iNOS‐ and ED2‐positive macrophages in the tongues from the AGVHD and control rats. Infiltrating macrophages in the lamina propria were counted. Cell numbers/mm² as mean ± standard deviation (s.d.). *Significantly different at P < 0·05 compared with iNOS‐positive cells in the control tongue and P < 0·01 compared with ED2 in the AGVHD tongue (Student's t‐test). Increasing ratio of M1 or M2 macrophages in the AGVHD‐tongue, relative to those in the control tongue. *Significantly different at P < 0·01(Student's t‐test). All experiments performed in quadruplicate.

Figure 3

Induction of laminin to activated macrophage migration. (a) Dual immunofluorescent images of laminin (red) and CD29 (green). Original magnification, ×200. (b) CD29‐positive cell migration was examined by a Transwell migration assay. Laminin was placed into the lower chamber well. The wells of the upper chamber received isolated ED1‐positive cells from the spleens and tongues of acute graft‐versus‐host disease (AGVHD) rats, pretreated with or without (control) an anti‐CD29 antibody. The cells were allowed to migrate towards laminin placed into the lower chamber of the Transwell system for 6, 12 and 18 h (h) at 37°C in a 5% CO₂ humidified atmosphere. The figure represents the results of five different experiments expressed as the mean ± standard deviation (s.d.). There are no significant differences in groups jointed to horizon bar. *Significantly different at P < 0·05 [analysis of variance (anova) followed by Scheffé's test]. All experiments performed in quintuplicate.

Figure 4

Loss of the epithelial basement membrane components mediated by MMP‐2. (a) Dual immunofluorescent images of laminin (red) and inducible nitric oxide synthase (iNOS) (green) in both early and advanced acute graft‐versus‐host disease (AGVHD) of the oral mucosa. Arrows indicate a loss of laminin expression on the basement membrane. Original magnification, ×200. (b) Basal immunofluorescence of matrix metalloproteinase (MMP)‐2 in the oral mucosa of untreated rats. The nucleus was stained with Hoechst 33324 (blue). (c) Using quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analyses, the tissue expression of MMP‐2 in tongues from untreated (grey) and AGVHD‐mediated (black) rats. Results are expressed as fold increases in mRNA expression (normalized to that of G3PDH mRNA) and compared with results for the oral mucosa from untreated rats. The results are presented as the mean ± standard deviation (s.d.) of five independent experiments. *Significantly different at P < 0·05 compared with the control rats (Student's t‐test). G3PDH = glyceraldehyde 3‐phosphate dehydrogenase. All experiments performed in quintuplicate. (d) Dual immunofluorescent images of MMP‐2 (red) and iNOS (green) in the AGVHD‐tongue. Nuclei were stained with Hoechst 33324 (blue). Original magnification, ×200. All experiments performed in quintuplicate.

Figure 5

Intra‐epithelial infiltration of activated macrophages mediated by the monocyte chemoattractant protein‐1 (MCP‐1) and C‐C chemokine receptor type 2 (CCR2) pathway. (a) Dual immunofluorescent images of iNOS (red) and MCP‐1 (green) in oral mucosal acute graft‐versus‐host disease (AGVHD). The nucleus was stained with Hoechst 33324. Original magnification, ×200. (b) Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) results for MCP‐1 and CCR2 mRNA expression in the oral mucosa from the untreated (grey) and AGVHD‐mediated (black). Results are the mRNA fold increases (normalized to G3PDH mRNA) in the oral mucosa from the AGVHD rats and compared with results from the untreated rats. Vertical lines represent the means ± standard deviation (s.d.) of five independent experiments, each performed in triplicate. *Significantly different at P < 0·05 compared with the control rats (Student's t‐test). G3PDH = glyceraldehyde 3‐phosphate dehydrogenase. All experiments performed in quintuplicate. (c) Adhesion of activated macrophages taken from spleens and tongues of AGVHD‐mediated rats to the oral epithelial cells by Stamper–Woodruff binding assay (SWBA). The pretreatment of macrophages examined the inhibition of macrophage binding to an anti‐iNOS or CCR2 antibody, as well as the pretreatment of oral epithelia with the anti‐MCP‐1 antibody. Results are mean ± standard deviation (s.d.) for five independent experiments. There are no significant differences in groups jointed to vertical bar. *Significantly different at P < 0·05 [analysis of variance (anova) followed by Scheffé's test). All experiments performed in quintuplicate.