Discoid lupus erythematosus (DLE) is characterized by features of disease activity such as erythema and scale and damage such as dyspigmentation and scarring [1]. Analogous changes histologically, such as inflammatory cells and dermal scarring, also occur. The inflammatory infiltrate consists of lymphocytes, macrophages, and plasmacytoid dendritic cells (pDCs) [2]. Because immunopathology studies in DLE have yet to subdivide DLE skin into separate phases, changes in the inflammatory infiltrate as DLE lesions transition from the inflammatory to scarring phase are unknown. Thus, we characterized the inflammatory infiltrate in different stages of DLE using immunohistochemistry. We hypothesized that changes in inflammatory cells occurred as DLE lesions develop dermal scarring. This would provide new insights into the pathophysiologic mechanisms behind the disease course of DLE.
Skin biopsies were retrospectively obtained from 30 DLE patients seen at the University of Texas Southwestern Medical Center and Parkland Health and Hospital System outpatient dermatology clinics between 2006 and 2016. This study was approved by the UT Southwestern Institutional Review Board and complied with Declaration of Helsinki. Patients included were not applying topical steroids or on systemic treatment for at least one month prior to biopsy. Immunostains for T cells (CD3, CD4, CD8), B cells (CD20), pDCs (CD123), macrophages (CD163), neutrophils (myeloperoxidase), and plasma cells (CD138) were performed according to established protocols [3]. Two independent observers (G.H. and B.F.C.) graded the overall intensity of the infiltrate of the most representative areas of three regions: interfollicular interface, perifollicular, and perivascular on a 0 to 3 scale (0: none, 1: mild, 2: moderate, 3: marked). Each immunostain was graded semi-quantitatively on a 0 to 3 scale based on percentage of positive-staining cells (0: <1%, 1: 1–25%, 2: 26–50%, 3: >50%). Median staining intensities for each immunostain in each region were calculated. Non-parametric two group comparisons were performed using the Mann-Whitney Test, with p-values <0.05 declared significant. All statistical analysis was performed using Graph-Pad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA).
Demographic and clinical information is available in Supplemental Table 1. We designated three stages of DLE based on the presence or absence of inflammation and/or dermal scarring: inflammatory DLE without (DLE-I, N = 9) and with dermal scarring (DLE-I/S, N = 18), inactive DLE with dermal scarring (DLE-S, N = 3). The DLE-S group consisted of end stage DLE lesions with scarring alopecia and marked dyspigmentation without any clinically apparent inflammation. Given the minimal inflammatory infiltrate present in DLE-S skin, we were unable to estimate and compare their relative percentages of positive immunostainings to the DLE-I and DLE-I/S groups.
CD3+ and CD4+ T cells were the predominant inflammatory cell in the interfollicular interface, perifollicular, and perivascular regions in most biopsy specimens, regardless of disease stage (Fig. 1a and b, Table 1). CD163+ macrophages were the next most common, followed by CD8+ T cells, CD20+ B cells and CD123+ pDCs. CD138+ plasma cells and neutrophils were infrequent. Median grades of cells staining positive in each region in DLE-I and DLE-I/S skin are summarized in Table 1. The limited inflammatory cell infiltrate in DLE-S skin primarily consisted of CD163+ macrophages and CD4+ T cells.
Fig. 1.
Composition of the inflammatory infiltrate in DLE-I and DLE-I/S. CD4+ lymphocytes were present in DLE-I skin (a) and DLE-I/S skin (b) at similar frequencies at the interfollicular interface, perifollicular, and perivascular regions. CD8+ lymphocytes were more frequently present in DLE-I skin (c) than DLE-I/S skin (d) in all three regions. CD20+ lymphocytes were less frequent in DLE-I skin (e) than DLE-I/S skin (f) in the interfollicular interface, perifollicular, and perivascular areas. Magnification: 40×. In summary (g), CD8+ lymphocytes (solid line) were increased in DLE-I skin. CD20+ B cells (dashed line) and the CD4:CD8 ratio (dotted line) were both increased in DLE-I/S skin. The mean overall grade is the mean of all three regions examined for a given immunostain.
Table 1.
Median grade of cells staining positive in specific regions of DLE skin.
| Interfollicular interface DLE-I |
DLE-I/S |
p-value | Perifollicular DLE-I |
DLE-I/S | p-value | Perivascular DLE-I |
DLE-I/S | p-value | |
|---|---|---|---|---|---|---|---|---|---|
| CD3 | 3 | 2.5 | 0.52 | 3 | 3 | 0.57 | 3 | 3 | 0.94 |
| CD4 | 2.5 | 2.75 | 0.84 | 3 | 3 | 0.72 | 2.5 | 3 | 0.34 |
| CD8 | 1.75 | 1 | 0.01 | 2 | 1 | <0.0001 | 2 | 1 | 0.02 |
| CD20 | 0.5 | 1 | 0.02 | 1 | 2 | <0.0001 | 1 | 1.75 | 0.0003 |
| CD123 | 0.75 | 1 | 0.16 | 1 | 1 | 0.35 | 1 | 1 | 0.33 |
| CD138 | 0 | 0.5 | 0.09 | 0.5 | 1 | 0.14 | 0.5 | 1 | 0.13 |
| CD163 | 2 | 2.5 | 0.25 | 1.5 | 1.5 | 0.30 | 1.5 | 1.5 | 0.78 |
| MPO | 0.5 | 0.5 | 0.34 | 0 | 0.25 | 0.42 | 0 | 0 | 0.49 |
| CD4:CD8 ratio | 2.5 | 4.25 | 0.07 | 1 | 6.75 | <0.0001 | 1.75 | 6.5 | 0.003 |
| Median intensity of inflitrate | 1.75 | 1 | 0.04 | 2 | 2.5 | 0.30 | 1.75 | 2 | 0.62 |
Abbreviations: DLE-I, inflammatory DLE without dermal scarring; DLE-I/S, inflammatory DLE with dermal scarring.
In comparing different phases of DLE, few differences in CD3+ and CD4+ cells were observed, but this was not unexpected given the complex nature of CD4+ T cells and their involvement in multiple (e.g. pro-inflammatory, pro-humoral, regulatory) immune pathways. In contrast, while CD8+ cells were a prominent component of the inflammatory infiltrate in DLE-I skin, they were less significantly frequent in DLE-I/S skin at the interfollicular interface (median: 1.75 vs. 1, p = 0.01), perifollicular regions (2 vs. 1, p <0.0001), and perivascular regions (2 vs. 1, p = 0.02) (Fig. 1c–d, Table 1). As a result, the CD4:CD8 ratio was significantly increased in DLE-I/S skin compared to DLE-I in both perifollicular (6.75 vs. 1, p <0.0001) and perivascular regions (6.5 vs. 1.75, p = 0.003) (Table 1). While some prior studies have reported CD8+ T cells to be the predominant T cell in DLE skin lesions [2,4], others have found equal proportions of CD4+ and CD8+ lymphocytes [5], or an abundance of CD4+ cells over CD8+ cells in DLE skin [6,7]. As we observed distinctive differences in the frequency of CD8+ T cells in early and late lesions, we speculate that the variation in previous results is due to differences in disease phases of DLE skin biopsies in these studies. The decrease in CD8+ T cells from DLE-I to DLE-I/S skin illustrates that CD8+ T cells play an important role in the initiation of DLE. CD8+ T cells produce serine proteases such as granzyme B that can induce keratinocyte apoptosis through caspase activation [5]. Apoptotic keratinocytes can amplify immune response because they are targeted by macrophages and autoantibodies, which are attracted by nuclear antigens displayed at the surface of these keratinocytes.
CD20+ B cells were less frequent in DLE-I than DLE-I/S skin at the interfollicular interface (0.5 vs. 1, p = 0.02), perifollicular areas (1 vs. 2, p <0.0001), and perivascular regions (1 vs. 1.75, p = 0.0003) (Fig. 1e–f, Table 1). Similarly, CD138+ plasma cells trended towards being more common in all three regions in DLE-I/S compared to DLE-I skin, including interfollicular interface, perifollicular regions, and perivascular regions, but did not reach statistical significance (Table 1). B cells have been previously shown to be increased in DLE skin lesions [8], and can secrete autoantibodies that perpetuate the immune response. The increase in B cells in more long-standing DLE-I/S skin lesions suggests that they may play a greater role in later findings in DLE such as scarring. B cells, via production of cytokines, can mediate fibrosis in the skin manifested by increased ollagen production [9] and fibroblast proliferation. Autoantibody production by B cells, such as anti-platelet-derived growth factor receptor antibodies, has also been linked to fibrosis [10]. In summary, the inflammatory infiltrate changes as lesions progress from early DLE-I lesions to later DLE-I/S and DLE-S lesions. CD8+ T cells are prominent in DLE-I lesions, while CD20+ B cells became a greater component of the inflammatory cell infiltrate in later DLE lesions. Our results provide evidence that the inflammatory process changes in different stages of DLE skin.
Acknowledgments
Conflicts of interest
Dr. Chong is an investigator for Daavlin Corporation and Biogen Incorporated, but these relationships are not relevant to the subject matter of this manuscript.
Funding/Support
The research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number K23AR061441, by the National Center for Advancing Translational Sciences of the National Institutes of Health under award Number TL1TR001105, and by the ProPath Dermatopathology Trainee Research Grant. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
We are indebted to Ms. Rose Ann Cannon for assisting in the manuscript preparation. We also would like to thank ProPath for their technical assistance in the immunohistochemical stainings.
Footnotes
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci.2016.12.004.
Contributor Information
Jack C. O’Brien, University of Texas Southwestern Medical Center Department of Dermatology, Dallas, TX, USA
Gregory A. Hosler, University of Texas Southwestern Medical Center Department of Dermatology, Dallas, TX, USA ProPath, Dallas, TX, USA.
Benjamin F. Chong, University of Texas Southwestern Medical Center Department of Dermatology, Dallas, TX, USA.
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