Inflammation and extracellular matrix degradation mediated by activated transcription factors nuclear factor-kappaB and activator protein-1 in inflammatory acne lesions in vivo

Abstract

Acne is the most common skin disease, causing significant psychosocial problems for those afflicted. Currently available agents for acne treatment, such as oral antibiotics and isotretinoin (Accutane), have limited use. Thus, development of novel agents to treat this disease is needed. However, the pathophysiology of acne inflammation is poorly understood. Before new therapeutic strategies can be devised, knowledge regarding molecular mechanisms of acne inflammation is required. We report here that transcription factors nuclear factor-kappaB and activator protein-1 are activated in acne lesions with consequent elevated expression of their target gene products, inflammatory cytokines and matrix-degrading metalloproteinases, respectively. These elevated gene products are molecular mediators of inflammation and collagen degradation in acne lesions in vivo. This new knowledge enables a rational strategy for development of pharmacological agents that can target the inflammation and matrix remodeling that occurs in severe acne.

Figure 1

Activation of NF-κB in skin specimens from inflammatory acne lesions. An inflammatory papule (B) and adjacent clinically normal skin (A) were obtained from acne patients. A component of NF-κB, p65 protein, was detected with biotinylated antibody in combination with Texas Red. p65 protein is stained red by this technique. A, inset, reveals cytoplasmic staining in a chicken-wire pattern in the epidermis, whereas B, inset, demonstrates a nuclear solid dot pattern. The specimens are from one subject and are representative of the findings in involved and uninvolved tissue from four patients.

Figure 2

Enhanced expression of inflammatory cytokines in inflammatory acne lesions. Skin was obtained from inflammatory papules and uninvolved areas of facial acne. Tissue specimens were assayed for cytokine mRNA levels using real-time quantitative RT-PCR. The values shown are means, with standard errors indicated by the bars. For each cytokine, mRNA levels were significantly higher in inflammatory acne than in uninvolved control skin (TNF-α, P < 0.05; IL-1β, P < 0.007; IL-8, P < 0.001; IL-10, P < 0.001; n = 8). ▪, uninvolved skin; □, acne lesion.

Figure 3

cJun, a component of AP-1 transcription factor, is induced in inflammatory acne lesions. An inflammatory papule (B) and adjacent uninvolved skin (A) were obtained from acne patients. A component of AP-1, cJun protein, was detected by peroxidase immunohistology. A: In uninvolved skin cJun expression is minimal. In inflammatory acne lesions, however, cJun staining is markedly intranuclear and prominent in follicular and perifollicular epidermis and in dermis (C is inset of B). The specimens are from one subject and are representative of the findings in involved and uninvolved tissue from eight patients.

Figure 4

Enhanced expression of AP-1 regulated MMPs in inflammatory acne lesions. Skin was obtained from inflammatory papules and uninvolved areas of facial acne. For MMP mRNA levels, real-time RT-PCR was used. The values shown are means, with standard errors indicated by the bars. A: For each MMP, mRNA levels were significantly higher in inflammatory acne than in uninvolved skin (MMP-1, P < 0.008; MMP-3, P < 0.006; MMP-9, P < 0.003; n = 6; ▪, uninvolved skin; □, acne lesion). MMP-1, -3, and -9 protein expression levels were assessed by immunohistology. B: Consistent with their mRNA data, the expression of three MMP proteins (revealed as brown staining) was increased in acne lesions as compared to uninvolved skin. Neutrophil collagenase (MMP-8) expression in inflammatory acne lesions was assessed with Western blot analyses. An inflammatory papule and adjacent uninvolved skin were obtained from three acne patients. C: Bands from the Western blot analyses are shown.

Figure 5

Induction of collagenolytic activity and increased degraded collagen levels in skin specimens from acne patients. Collagenase activity in inflammatory acne (B) and uninvolved skin (A) was measured with the use of fluorescein-labeled collagen as substrate. The specimens are from one subject and are representative of the findings in tissue from three patients. The green color is fluorescein-labeled collagen that was coated onto a glass slide. The skin section was laid on top of the slide and incubated for 24 hours to allow collagenase in the tissue to degrade the fluorescein-labeled collagen on the slide. Darkened areas, especially noticeable in B (asterisks) are due to the degradation of colored substrate by collagenase. For C skin was obtained from inflammatory papules and uninvolved areas of facial acne. α-Chymotrypsin-sensitive degraded/fragmented collagen was expressed as a percentage of total skin collagen (ratio of hydroxyproline levels). *P < 0.04; n = 5; ▪, uninvolved skin; □, acne lesion.

Figure 6

Type I and type III procollagen synthesis is induced in inflammatory acne lesions. An inflammatory papule and adjacent uninvolved skin were obtained from an acne patient. A: Tissue specimens were assayed for type I and type III procollagen mRNA levels using real-time quantitative RT-PCR. Data are expressed as fold greater than the values of uninvolved skin. 36B4 mRNA was used to normalize the expression level of other genes. Immunostaining of procollagen I in uninvolved and involved skin was performed with SP.1D (B) and PIC antibodies (C). Positively stained dermal fibroblasts are much more numerous in the inflammatory acne lesion, as compared to the uninvolved skin.

Figure 7

Hypothetical model of the pathophysiology of inflammatory acne and dermal damage. In inflammatory acne lesions, NF-κB signaling is activated. As a consequence, NF-κB-driven inflammatory cytokine genes (eg, TNF-α and IL-1β) are induced. These primary cytokines will propagate the inflammatory response by acting on endothelial cells to elaborate adhesion molecules (eg, ICAM-1) to facilitate recruitment of inflammatory cells into the skin. TNF-α and IL-1β will also stimulate the production of secondary cytokines, such as IL-8, which can aid in chemotaxis of inflammatory cells. By working through their cell surface receptors, TNF-α and IL-1β not only amplify the NF-κB signaling cascade, but also activate MAP kinases to stimulate AP-1-mediated gene transcription. As a consequence of AP-1 activation (cJun induction), AP-1-driven MMPs are elaborated by resident skin cells. Along with MMP-8 and neutrophil elastase brought in by PMNs, they degrade the matrix. This is followed by matrix synthesis and repair, which is imperfect. Most of the imperfections would leave clinically undetectable deficits in the organization or composition, or both, of the extracellular matrix. However, when they occur to a significant extent throughout time, accompanied by sustained procollagen synthesis, acne scarring becomes clinically visible.