Evolution of the Macrophage CD163 Phenotype and Cytokine Profiles in a Human Model of Resolving Inflammation - PubMed (original) (raw)

Evolution of the Macrophage CD163 Phenotype and Cytokine Profiles in a Human Model of Resolving Inflammation

Betsy J Evans et al. Int J Inflam. 2013.

Abstract

Cantharidin skin blisters were examined over two days to model the acute and resolving phases of inflammation in human skin. Four blisters were created by topical administration of cantharidin (0.1% v/v) to the forearm of healthy volunteers, with IRB approval. Duplicate skin blisters were aspirated at 16 and 40 hours to model the proinflammatory and resolving phases, respectively. There was a significant increase in leukocyte infiltrate at 40 h with appearance of a "resolving macrophage" phenotype CD14(+)CD163(+) by flow cytometry. Neutrophils acquired apoptotic markers at 40 h and were observed to be phagocytosed by macrophagic "Reiter's" cells. Multiplex cytokine analysis demonstrated that monocyte chemoattractant protein (MCP-1/CCL2), interleukin- (IL-) 6, IL-8/CXCL8, macrophage inflammatory protein (MIP1 α /CCL3), MIP-1 β /CCL4, tumor necrosis factor- (TNF-) α , and eotaxin (CCL11) were all significantly upregulated at 16 h compared with 40 h. In contrast, immunoregulatory transforming growth factor- (TGF-) β , macrophage-derived chemokine (MDC/CCL22), and interferon-inducible protein (IP-10/CXCL10) were significantly elevated at 40 h. Our results demonstrate that the phases of inflammation and resolution can be discriminated in a two-day model of dermal wound healing. This confirms and extends our understanding of wound repair in humans and provides a powerful research tool for use in clinical settings and to track the molecular benefits of therapeutic intervention.

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Figures

Figure 1

Figure 1

Leukocyte infiltration into cantharidin-induced skin blisters. Quadruplicate skin blisters were established by topical application of cantharidin (0.1%) to the forearm of 10 healthy individuals. Blister fluid was collected from duplicate blisters at 16 h and 40 h, respectively, and analyzed as follows (a) Fluid volume (mL) and cellularity (×105). Each point represent the average from 2 skin blisters; horizontal lines represents the population mean. (b) Differential leukocyte subpopulations. PMN: polymorphonuclear phagocytes; EOS: eosinophils; LYMPH: lymphocytes; MONO: monocytes/macrophages.

Figure 2

Figure 2

Photomicrographs of blister fluid at (a) 16 hours and (b) 40 hours. Granulocytes reveal morphological changes characteristic of apoptosis at 40 hours, including vacuolation of cytoplasm, size shrinkage, and engulfment by phagocytic “Reiter's” cells (arrow). Magnification = 100x.

Figure 3

Figure 3

Flow cytometric analysis of PMNs in blister fluid. (a) Forward and side-scatter analyses reveal a subpopulation of smaller, less granular PMN. Gating on this subpopulation demonstrates expression of apoptosis markers, Annexin V, and Propidium Iodide (PI). Annexin V+ cells are considered at an early stage of apoptosis, Annexin V+/PI+ cells are at an intermediate stage, and Annexin V−/PI+ cells at a late stage of apoptosis. No expression of apoptosis markers is seen in the viable cell gate. (b) PMNs at 40 hours demonstrate an increased proportion of cells exhibiting a CD16low phenotype, also characteristic of apoptotic cells.

Figure 4

Figure 4

Enzyme-linked immunosorbent assay (ELISA) detection of inflammatory regulators in blister fluid: (a) MCP-1/CCL2, (b) IL-8/CXCL8, (c) MDC/CCL22, and (d) TGF_β_. Blister fluid from n = 10 individuals was analyzed by ELISA in duplicate, at each of the timepoints. Results are expressed as mean ± S.E.M. P values represent statistical differences between 16-hour and 40-hour timepoints analyzed by unpaired t test.

Figure 5

Figure 5

Multiplex bead array (Luminex) analysis of inflammatory regulators in blister fluid: (a) MCP-1/CCL2, (b) IL-8/CXCL8, (c) IL-6, (d) TNF_α_, (e) MIP-1_α_/CCL3, (f) MIP-1_β_/CCL4, (g) Eotaxin/CCL11, and (h) IP-10/CXCL10. Blister fluid from n = 10 individuals was analyzed by multiplex bead array at each of the timepoints. Results are expressed as mean ± S.D. P values represent statistical differences between 16-hour and 40-hour timepoints analyzed by unpaired t test.

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