Role of inflammatory cells, cytokines and matrix metalloproteinases in neutrophil-mediated skin diseases

Abstract

Pyoderma gangrenosum (PG) is a rare, immune-mediated inflammatory skin disease presenting with painful ulcers having undermined edges. Less commonly, bullous and vegetative variants exist. Histology consists of a neutrophil-rich dermal infiltrate. We characterized immunohistochemically the infiltrate in different variants of PG and in another neutrophilic dermatosis as Sweet's syndrome. We studied 21 patients with PG, eight with Sweet's syndrome and 20 controls, evaluating skin immunoreactivity for inflammatory cell markers (CD3, CD163 and myeloperoxidase), cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-8 and IL-17], metalloproteinases (MMP-2 and MMP-9) and vascular endothelial growth factor (VEGF). Immunoreactivities of CD3, CD163, myeloperoxidase, TNF-α, IL-8, IL-17, MMP-2, MMP-9 and VEGF were significantly higher in both PG and Sweet's syndrome than in controls (P=0·0001). Myeloperoxidase (neutrophil marker), IL-8 (cytokine chemotactic for neutrophils) and MMP-9 (proteinase-mediating tissue damage) were expressed more significantly in both ulcerative and bullous PG than in vegetative PG as well as in Sweet's syndrome (P=0·008-P=0·0001). In ulcerative PG, the expression of CD3 (panT cell marker) and CD163 (macrophage marker) were significantly higher in wound edge than wound bed (P=0·0001). In contrast, the neutrophil marker myeloperoxidase was expressed more significantly in wound bed than wound edge (P=0·0001). Our study identifies PG as a paradigm of neutrophil-mediated inflammation, with proinflammatory cytokines/chemokines and MMPs acting as important effectors for the tissue damage, particularly in ulcerative and bullous PG where damage is stronger. In ulcerative PG, the wound bed is the site of neutrophil-recruitment, whereas in the wound edge activated T lymphocytes and macrophages pave the way to ulcer formation.

Fig. 1

Immunohistochemical reactivity of inflammatory cell markers (CD3, CD163 and myeloperoxidase), cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-8 and IL-17], metalloproteinases (MMP-2 and MMP-9) and vascular endothelial growth factor (VEGF) in different variants of pyoderma gangrenosum, i.e. ulcerative (UPG), vegetative (VPG) and bullous (BPG), in classic Sweet's syndrome and in 20 normal controls. Immunoreactivity was scored according to the number of immunoreactive cells per field (×200): 0 = no immunoreactive cells; 1 = 1–5 cells; 2 = 6–20 cells; 3 = >20 cells. The expression of the above-mentioned effector markers was significantly higher in both PG and Sweet's syndrome than in normal controls (P = 0·0001). Myeloperoxidase (neutrophil marker), IL-8 (cytokine chemotactic for neutrophils) and MMP-9 (proteinase-mediating tissue damage) were expressed more significantly in both ulcerative and bullous pyoderma gangrenosum (PG) than in vegetative PG as well as in Sweet's syndrome (P = 0·008–P = 0·0001). In contrast, the macrophage marker CD163 was expressed more significantly in vegetative PG than in the other PG variants as well as in Sweet's syndrome (P = 0·032–P = 0·003). The expression of TNF-α or IL-17 was not significantly different within the PG variants as well as between PG and Sweet's syndrome. VEGF values were also not significantly different in the PG variants and Sweet's syndrome.

Fig. 2

Immunohistochemical reactivity of inflammatory cell markers (CD3, CD163 and myeloperoxidase), cytokines (TNF-α, IL-8 and IL-17), metalloproteinases (MMP-2 and MMP-9) and vascular endothelial growth factor (VEGF) in ulcerative pyoderma gangrenosum at the wound edge and wound bed. Immunoreactivity was scored according to the number of immunoreactive cells per field (×200): 0 = no immunoreactive cells; 1 = 1–5 cells; 2 = 6–20 cells; 3 = >20 cells. The expression of both CD3 (a panT cell marker and CD163 (a macrophage marker) were significantly higher in wound edge than in wound bed (P = 0·0001 for both). In contrast, the neutrophil marker myeloperoxidase was expressed more significantly in the wound bed than in the wound edge (P = 0·0001). Also, the chemokine IL-8 (P = 0·0001) and the matrix metalloproteinases (MMP) 2 (P = 0·009) and 9 (P = 0·001) showed a significantly higher immunoreactivity in the wound bed than in the wound edge. In the wound edge the immunoreactivity for both TNF-α and IL-17 was slightly increased (P = 0·017 and P = 0·028, respectively). VEGF values were not significantly different in the wound edge and bed.

Fig. 3

Immunohistochemical studies of CD3, CD163 and myeloperoxidase in ulcerative pyoderma gangrenosum (PG) (original magnification ×100). An intense expression of both CD3 (panT-cell marker) and CD163 (macrophage antigen) is evident at the wound edge (upper panels), where only slight reactivity for the neutrophil marker myeloperoxidase is seen. In contrast, at the wound bed (lower panels), the inflammatory infiltrate is slightly immunostained for CD3 and CD163, with strong expression of myeloperoxidase.

Fig. 4

Immunohistochemical studies of CD3, CD163 and myeloperoxidase in vegetative pyoderma gangrenosum (PG) (upper panels) and bullous PG (lower panels) (original magnification ×100). In vegetative PG, an abundant expression of CD163 with only mild staining for both CD3 and myeloperoxidase is evident. The inflammatory infiltrate of bullous PG is characterized by very strong immunoreactivity for myeloperoxidase and slight expression of both CD3 and CD163.

Fig. 5

Immunohistochemical studies of CD3, CD163 and myeloperoxidase in Sweet's syndrome (original magnification ×100). A moderate focal expression of CD3 and a slight expression of both CD163 and myeloperoxidase are evident.

Fig. 6

Immunohistochemical studies of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in ulcerative pyoderma gangrenosum (PG) (original magnification ×100). The immnoreactivity of IL-8 is strong at the wound bed. VEGF is moderate and diffuse.

Fig. 7

Immunohistochemical studies of metalloproteinase (MMP)-9 in ulcerative pyoderma gangrenosum (PG) (wound bed), vegetative PG and bullous PG (original magnification ×100). A strong MMP-9 expression is observed in both ulcerative and bullous PG, whereas only slight immunostaining is seen in vegetative PG.

Fig. 8

Immunohistochemical studies of metalloproteinase (MMP)-2 and MMP-9 in classic Sweet's syndrome (original magnification ×100). The reactivity of both MMP-2 and MMP-9 is low.