Association of low numbers of CD206-positive cells with loss of ICC in the gastric body of patients with diabetic gastroparesis

Abstract

Background: There is increasing evidence for specific cellular changes in the stomach of patients with diabetic (DG) and idiopathic (IG) gastroparesis. The most significant findings are loss of interstitial cells of Cajal (ICC), neuronal abnormalities, and an immune cellular infiltrate. Studies done in diabetic mice have shown a cytoprotective effect of CD206+ M2 macrophages. To quantify overall immune cellular infiltrate, identify macrophage populations, and quantify CD206+ and iNOS+ cells. To investigate associations between cellular phenotypes and ICC.

Methods: Full thickness gastric body biopsies were obtained from non-diabetic controls (C), diabetic controls (DC), DG, and IG patients. Sections were labeled for CD45, CD206, Kit, iNOS, and putative human macrophage markers (HAM56, CD68, and EMR1). Immunoreactive cells were quantified from the circular muscle layer.

Key results: Significantly fewer ICC were detected in DG and IG tissues, but there were no differences in the numbers of cells immunoreactive for other markers between patient groups. There was a significant correlation between the number of CD206+ cells and ICC in DG and DC patients, but not in C and IG and a significant correlation between iNOS+ cells and ICC in the DC group, but not the other groups. CD68 and HAM56 reliably labeled the same cell populations, but EMR1 labeled other cell types.

Conclusions & inferences: Depletion of ICC and correlation with changes in CD206+ cell numbers in DC and DG patients suggests that in humans, like mice, CD206+ macrophages may play a cytoprotective role in diabetes. These findings may lead to novel therapeutic options, targeting alternatively activated macrophages.

Keywords: gastroparesis; interstitial cells of Cajal; macrophages.

Figure 1

Significantly fewer ICC were detected in the circular muscle layer of Idiopathic Gastroparesis (IG, 2.1±0.5 cells/field) and Diabetic Gastroparesis (DG, 1.67±0.6 cells/field) compared to Controls (C, 4.95±1.2 cells/field) and Diabetic Controls (DC, 3.8±0.72 cells/field). Data show average values for counts from 39 fields from each patient as points, population means ± SEM shown as whiskers, n = 10 patients for each group, * indicates P < 0.05, one way ANOVA with Newman Keuls post test.

Figure 2

Representative images for CD45 immunoreactivity in the gastric circular muscle layer from (A) Control (B) Diabetic Control (C) Idiopathic Gastroparesis (D) Diabetic Gastroparesis Scale bar = 100μM (E) Number of CD45 positive cells was not different between the four groups. Data show average values for counts from 39 fields from each patient as points, population means ± SEM shown whiskers, n = 10 patients for each group, P >0.05, One way ANOVA.

Figure 3

No correlation between number of CD45 positive cells and ICC (A) Control: r² = 0.111, Slope=0.536±0.538; (B) Diabetic Control: r² = 0.335, Slope=0.209±0.104; (C) Idiopathic Gastroparesis: r² =0.3215, Slope= -0.241±0.124; (D) Diabetic Gastroparesis: r² =0.008, Slope=0.062±0.238. Each point represents the data from a single patient and are means ± SEM for counts from 39 fields. Data were fit by linear regression using Graphpad Prism. Dark line shows the fit, Grey dotted lines show the 95% confidence limits for the fit.

Figure 4

Representative images of macrophages markers (A) CD68 (B) HAM56 (C) Merged image (CD68 green) and (HAM56 red) in the circular muscle layer from the gastric body. White arrowhead shows CD68 positive, HAM56 negative cells. White arrow shows CD68 negative, HAM56 positive cells. Scale bar = 100μM

Figure 5

Representative images for CD206 immunoreactivity in the gastric circular muscle layer from (A) Control (B) Diabetic Control (C) Idiopathic Gastroparesis (D) Diabetic Gastroparesis. Scale bar = 100μM (E) Number of CD206 positive cells was not different between the four groups. Data show average values for counts from 39 fields from each patient as points, population means ± SEM shown whiskers, n = 10 patients for each group, P >0.05, One way ANOVA.

Figure 6

Correlation between number of CD206 positive cells and ICC. There was no correlation between CD206 positive cells and ICC in (A) Controls and (C) Idiopathic Gastroparesis. There was a significant correlation between CD206 positive cells and ICC in (B) Diabetic Controls (P=0.0285) and (D) Diabetic Gastroparesis (P=0.001). Each point represents the data from a single patient and are means ± SEM for counts from 39 fields. Data were fit by linear regression using Graphpad Prism. Dark line shows the fit, Grey dotted lines show the 95% confidence limits for the fit.

Figure 7

Representative images for iNOS immunoreactivity in the gastric circular muscle layer from (A) Control (B) Diabetic Control (C) Idiopathic Gastroparesis (D) Diabetic Gastroparesis. (E) Number of iNOS positive cells is not different between the four groups. Data show average values for counts from 39 fields from each patient as points, population means ± SEM shown whiskers, n = 10 patients for each group, (P>0.05, One way ANOVA).

Figure 8

Correlation between number iNOS positive cells and ICC. There was no significant correlation between iNOS positive cells and ICC in (A) Controls (C) Idiopathic Gastroparesis and (D) Diabetic Gastroparesis. There was a correlation between number iNOS positive cells and ICC in (B) Diabetic Controls (P=0.0012). Each point represents the data from a single patient and are means ± SEM for counts from 39 fields. Data were fit by linear regression using Graphpad Prism. Dark line shows the fit, Grey dotted lines show the 95% confidence limits for the fit.