An abnormality in glucocorticoid receptor expression differentiates steroid responders from nonresponders in keloid disease

Abstract

Background: Glucocorticoids (GCs) are first-line treatment for keloid disease (KD) but are limited by high incidence of resistance, recurrence and undesirable side-effects. Identifying patient responsiveness early could guide therapy.

Methods: Nineteen patients with KD were recruited at week 0 (before treatment) and received intralesional steroids. At weeks 0, 2 and 4, noninvasive imaging and biopsies were performed. Responsiveness was determined by clinical response and a significant reduction in vascular perfusion following steroid treatment, using full-field laser perfusion imaging (FLPI). Responsiveness was also evaluated using (i) spectrophotometric intracutaneous analysis to quantify changes in collagen and melanin and (ii) histology to identify changes in epidermal thickness and glycosaminoglycan (GAG) expression. Biopsies were used to quantify changes in glucocorticoid receptor (GR) expression using quantitative reverse transcriptase polymerase chain reaction, immunoblotting and immunohistochemistry.

Results: At week 2, the FLPI was used to separate patients into steroid responsive (n = 12) and nonresponsive groups (n = 7). All patients demonstrated a significant decrease in GAG at week 2 (P < 0.05). At week 4, responsive patients exhibited significant reduction in melanin, GAG, epidermal thickness (all P < 0.05) and a continued reduction in perfusion (P < 0.001) compared with nonresponders. Steroid-responsive patients had increased GR expression at baseline and showed autoregulation of GR compared with nonresponders, who showed no change in GR transcription or protein.

Conclusions: This is the first demonstration that keloid response to steroids can be measured objectively using noninvasive imaging. FLPI is a potentially reliable tool to stratify KD responsiveness. Altered GR expression may be the mechanism gating therapeutic response.

Figure 1

(a) Experimental design. qRT‐PCR, quantitative reverse transcriptase polymerase chain reaction; GR IHC, glucocorticoid receptor immunohistochemistry; GAG, glycosaminoglycan. (b) Patient demographics. M, male; F, female.

Figure 2

Flux profiling to stratify patient responses to steroid treatment. Keloids from 19 patients were imaged using full‐field laser perfusion imaging. In each case, perfusion of keloid scar was normalized to the surrounding normal tissue. (a) Representative flux profiles are shown. Areas of high flux are red and low flux are blue. (b) Steroid‐dependent change in perfusion at week 2. Those who demonstrated reductions (below the line) in flux were designated responsive patients (RPs) and those who did not (above the line) were designated nonresponsive patients (nRPs). (c) Perfusion at week 0 for RPs (n = 12) and nRPs (n = 7). (d) Perfusion at week 4 for RPs and nRPs. *P < 0·05.

Figure 3

Clinical response to intralesional steroid injections. (a) Representative photographs of keloid scars of responsive patients (RPs) and nonresponsive patients (nRPs) at week 0 and week 4. Scale bar 2 cm. (b–e) Spectrophotometric intracutaneous analysis was used to quantify collagen and melanin chromophores. (b) Collagen chromophore at week 0 for RPs (n = 12) and nRPs (n = 7). (c) Steroid‐dependent change in collagen chromophore at weeks 2 and 4. (d) Melanin chromophore at week 0 for RP (n = 12) and nRP (n = 7). (e) Steroid‐dependent fold change in melanin chromophore at weeks 2 and 4. *P < 0·05.

Figure 4

Histological measurement of epidermal thickness and glycosaminoglycan (GAG) content. (a, b) Alcian blue staining was used to quantify GAGs. (a) GAG content at week 0 for responsive patients (RPs) (n = 12) and nonresponsive patients (nRPs) (n = 7). (b) Steroid‐dependent change in GAG content at weeks 2 and 4. (c–e) Sections were stained with haematoxylin and eosin, and epidermal thickness quantified at 10 different positions across each section. (c) Representative images are shown. White line indicates epidermal thickness. (d) Epidermal thickness at week 0 for RPs (n = 12) and nRPs (n = 7). (e) Steroid‐dependent change in epidermal thickness at weeks 2 and 4. *P < 0·05.

Figure 5

Immunohistological analysis of glucocorticoid receptor (GR) expression and localization. (a–d) Sections were immune‐labelled with a GR‐specific antibody (brown) and the nuclei counterstained (pink). (a) Representative images from both groups are shown. Original magnification ×20; E, epidermis; PD, papillary dermis; RD, reticular dermis. (b) GR expression at week 0 for responsive patients (n = 12) and nonresponsive patients (n = 7). (c) Steroid‐dependent change in GR expression at weeks 2 and 4. (d) Subcellular GR staining in the epidermis. Higher magnification (×40) images are shown below. Asterisks indicate nuclear GR localization. Arrows indicate immune cell infiltration. *P < 0·05.

Figure 6

Quantification of glucocorticoid receptor (GR) transcription and protein. (a, b, f) GR transcription was measured by quantitative polymerase chain reaction. (a) GR transcription at week 0 for responsive patients (RPs) (n = 12) and nonresponsive patients (nRPs) (n = 7). (b) Steroid‐dependent change in GR transcription at weeks 2 and 4. (c–d) GR protein expression was measured by immunoblotting using a GR‐specific antibody. (c) A representative immunoblot is shown. Tubulin is shown as a loading control. (d) GR expression at week 0 for RPs (n = 12) and nRPs (n = 7). (e) Steroid‐dependent change in GR expression at weeks 2 and 4. (f) GR transcription in keloid (n = 19) and nonkeloid scars (n = 5). *P < 0·05.