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The features of skin inflammation induced by lupus serum
Clinical Immunology 165 (2016) 4–11
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Clinical Immunology journal homepage: www.elsevier.com/locate/yclim
The features of skin inflammation induced by lupus serum Lena Liu b, Guangqion Xu a, Hui Dou a, Guo-Min Deng a,b,c,⁎ a b c
Key Lab of Antibody Techniques of Ministry of Health, Nanjing Medical University, Nanjing, China Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
a r t i c l e
i n f o
Article history: Received 16 February 2016 accepted with revision 17 February 2016 Available online 18 February 2016 Keywords: Systemic lupus erythematosus Lupus serum IgG Skin inflammation
a b s t r a c t We recently developed a model of lupus serum-induced skin inflammation, which was used to study the pathogenesis of skin injury in systemic lupus erythematosus (SLE). We further characterized the features of lupus serum-induced skin inflammation. This skin inflammation was evident within 3 h and lasted for at least two weeks. The skin inflammation was characterized by an influx of monocytic, CD11b + cells and by a scarcity of T and B lymphocytes. Depletion of IgG from the serum abrogated the skin inflammatory response. The skin inflammation was related to lupus patients' skin history but not to SLE disease activity and type of autoantibody. The expression of TNFR1, NF-kB and MCP-1 was increased locally in skin lesions. The TLR9 ligand and lupus serum act synergistically to trigger skin inflammation. These findings suggest that this novel model is valuable for the study of the pathogenesis and therapy of skin injury in SLE. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by high levels of autoantibody and multi-organ tissue damage, including kidney and skin damage [1,2]. Skin injury is second most common clinical manifestation in patients with SLE [3,4]. The molecular and cellular mechanisms involved in the expression of cutaneous LE remain unclear [5]. The skin injury of SLE can be divided into lupus erythematous (LE)specific or LE-nonspecific manifestations by histologic analysis of biopsy specimens [6]. The LE-nonspecific manifestations are commonly associated with systemic organ manifestations or other autoimmune diseases [6]. The LE-specific cutaneous manifestations may present without or with less severe systemic organ involvement and are further classified into several subtypes, including acute (A) CLE, subacute (S) CLE, chronic (C) CLE and intermittent (I) CLE, which was proposed to be a separate category for LE tumidus in 2004 and ICLE is not universally accepted [7–9]. ACLE, SCLE and CCLE are three common clinical subtypes of cutaneous LE. Localized ACLE is often referred to as the ‘malar rash’ or ‘butterfly rash’ of SLE, whereas generalized ACLE is frequently referred to as the ‘SLE rash’ [9]. The skin injuries share the following features of a lichenoid tissue reaction: hyperkeratosis; epidermal atrophy; liquefactive degeneration of the epidermal basal-cell layer; mononuclear cell infiltrates focused at the dermo-epidermal junction, perivascular areas and perifollicular areas; thickening of the basal membrane; and melanin pigment incontinence. ACLE typically presents abruptly in the context of a systemic disease, and almost all patients ⁎ Corresponding author at: Nanjing Medical University, China. E-mail address: [email protected] (G.-M. Deng).
http://dx.doi.org/10.1016/j.clim.2016.02.007 1521-6616/© 2016 Elsevier Inc. All rights reserved.
develop SLE [5]. ICLE seems to be a purely dermatological disease [10, 11]. The deposition of immune complexes containing IgG, IgM and complement (C) 3 is typically observed at the dermo-epidermal junction and is defined as a positive ‘lupus band’ test [12,13]. The production of autoantibodies directed against nuclear antigens and a myriad of other autoantigens characterizes SLE. In the skin, the invariably observed keratinocyte apoptosis may result in the cell surface expression and release of self-antigens, including DNA, Ro/SSA, La/SSB and histones [2,5]. Although exposure to UV and other environmental triggers may contribute to the initiation of cutaneous LE by triggering the apoptosis of keratinocytes, it is still unclear how the inflammatory process begins and is sustained [2,5]. IFN-α plays an important role in the pathogenesis of lupus erythematosus skin lesions [14]. Large numbers of IFN-producing plasmacytoid dendritic cells are also found in SLE skin lesions [15,16]. Tissue damage in SLE is associated with autoantibody production and immune complex formation and deposition [2]. An anti-doublestranded DNA antibody has been linked to kidney [2] and brain pathology [17]. Anti-acidic ribosomal protein P0 (anti-RPLP0) and antigalectin 3 antibodies are related to the development of skin lesions in SLE [18]. Ro52 is a common target of circulating autoantibodies in SLE. Ro52 is highly expressed in spontaneous and UV-induced cutaneous inflammation in CLE; approximately 80% of cells within infiltrates of CLE lesions are positively stained for Ro52 in the dermis [19]. The loss of autoantigen Ro52 induces skin inflammation and systemic autoimmunity [20]. This result indicates that Ro52 exerts an important regulatory role in lupus skin lesions. Apparently, Autoantigens bind to keratinocytes undergoing apoptosis and contribute to the inflammatory process [2]. MRL/lpr mice are a common animal model of SLE and spontaneously develop lupus-like clinical manifestations characterized by
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high levels of autoantibodies and multiorgan tissue damage, including skin injury [21,22]. These observations indicate that autoantibodies may play an important role in the expression of cutaneous lesions in SLE and led us to hypothesize that lupus serum induces skin inflammation. Recently, we have confirmed the hypothesis that serum from lupus patients and lupus-prone mice induced skin inflammation [23]. We also found that an animal model of lupus serum-induced skin inflammation is a valuable tool to investigate the pathogenesis and therapy of skin injuries in SLE [24,25]. In this paper, we further defined the features of skin inflammation induced by lupus serum.
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2.3. Histopathological examination Histopathological examination of skin was performed after routine fixation and paraffin embedding of the tissue. Tissue sections from the skin were cut and stained with hematoxylin and eosin. All slides were coded and evaluated in a ‘blinded to sample’ identity manner. The severity of skin inflammation was scored 0–4 as follows: grade 0, normal; grade 1, hyperplasia of epidermis; and grades 2–4, different amounts of infiltrating inflammatory cells in the skin with or without hyperplasia of epidermis [24]. 2.4. Immunohistochemical examination
2. Materials and methods 2.1. Mice and sera C57BL/6, BALB/c, Swiss and MRL/lpr mice were purchased from JAX Labs (Cold Harbor, USA). All mice were housed in the animal facility of the Beth Israel Deaconess Medical Center and Nanjing Medical University. Sera were collected from patients with SLE and MRL/lpr mice at different ages and normal C57BL/6 mice. Animal and human use protocols were approved by appropriate Beth Israel Deaconess Medical Center committees and by Nanjing Medical University committees. Sera were collected from patients with SLE and controls from Beth Israel Deaconess Medical Center and Jiangsu Province Hospital. Eighteen patients fulfilling ≥4 of 11 revised criteria of the American College of Rheumatology for the classification of SLE were studied. All patients had SLE disease-activity index (SLEDAI) scores ranging from 0 to 12. Medications were discontinued ≥24 h before venipuncture. Ten normal women served as controls in this study. Sera were collected from MRL/ lpr mice of different ages and normal C57BL/6 mice.
2.2. Injection protocol procedures Different doses of lupus serum were injected intradermally in the back of the neck of mice of different strains. All of the intradermal injections were performed on anesthetized mice [24].
After deparaffinization and antigen retrieval, samples were stained with antibodies to CD4 + (GK1.5), CD8 + (53.6.7), NF-κB and MCP-1 (Santa Cruz, CA) followed by incubation with biotinylated secondary antibodies, avidin-biotin-peroxidase complexes and 3-amino-9-ethylcarbazole containing H2O2. All sections were counterstained with Mayer's hematoxylin [24]. 2.5. Immunoglobulins Serum levels of IgG were measured by an enzyme-linked immunosorbent assay (ELISA). Antisera and immunoglobulin standards specific for IgG were purchased from Sigma. 2.6. Autoantibodies Serum antibody levels to denatured or single-stranded DNA (ssDNA) and to native or double-stranded DNA (dsDNA), were measured by ELISA using methylated bovine serum albumin (10 μg/ml) to precoat the wells, followed by coating with 50 μg of heat-denatured (boiled for 20 m and then cooled rapidly on ice) calf thymus DNA (Sigma) per milliliter or native calf thymus DNA, as previously described [23]. 2.7. Cytokine levels The cytokine level in mouse serum was determined by ELISA.
Fig. 1. Lupus serum induces skin inflammation. (A) Representative histopathological photomicrograph. Severity of skin inflammation of C57BL/6 mice sacrificed 3 d after intradermal inoculation of serum (100 μl) from a lupus patient with skin disease, a healthy control and PBS. (B) The severity of skin inflammation of LPS-responding C3H/HeN and LPSnonresponding C3H/HeJ mice 3 d after intradermal inoculation of lupus serum (100 μl) (n = 5 per group). (C) The severity of skin inflammation in C57BL/6 mice (n = 5 per group) with intradermal injection of 100 μl of serum from MRL/lpr mice and C57BL/6 mice at 16 weeks of age. (D) The severity of skin inflammation in C57BL/6 mice (n = 5 per group) with intradermal injection (ID), intra-venous injection or intraperitoneal injection of 100 μl of lupus serum. (E) Incidence of skin inflammation in various mouse strains with intradermal injection of 100 μl of serum from lupus patients. *p b 0.05; **p N 0.05.
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2.8. Statistical analysis Statistical evaluations were analyzed using Student's t-test. p ≤ 0.05 was considered statistically significant.
3. Results 3.1. Induction of skin inflammation by lupus serum Because IgG deposited in the skin of patients with SLE and lupusprone mice comes from blood circulation, we hypothesized that lupus serum containing IgG might induce skin inflammation. We injected PBS and serum from patients with SLE and healthy individuals directly into murine skin. Histopathological examination demonstrated that skin inflammation developed in mice with intradermal injection of serum from patients with SLE but not from healthy individuals and PBS (Fig. 1A). This result indicates that lupus serum can induce skin inflammation and that the skin inflammation was not caused by the surgical procedure because the skin inflammation did not develop in mice with intradermal injection of healthy control serum and PBS. Because skin inflammation induced by lupus serum might be caused by LPS contamination in lupus serum, we performed further investigations to exclude this possibility. To this aim, we injected lupus serum into the skin of LPS-nonresponder C3H/HeJ mice and LPS-resistant C3H/HeJ mice [26]. The results demonstrated that there was no difference in the severity and incidence of skin inflammation upon injection of lupus serum into LPS-resistant strain C3H/HeJ mice or their congenic LPS-responder strain C3H/HeN mice (Fig. 1B); in contrast, intradermal injection of LPS induced skin inflammation in C3H/HeN mice but not in C3H/HeJ mice (Fig. 1B). This result indicates that the induction of
skin inflammation was due to lupus serum rather than LPS contamination. To further define that skin inflammation is caused by lupus serum, we determined whether serum from lupus-prone mice induces skin inflammation. To this aim, we intradermally (ID) injected serum from lupus MRL/lpr mice and C57BL/6 mice at 16 weeks of age into the skin of normal C57BL/6 mice. We found that skin inflammation developed in mice injected with serum from lupus MRL/lpr mice but not from C57BL/6 mice (Fig. 1C). To further assess whether systemic administration of lupus serum might induce skin inflammation, we treated mice with intraperitoneal (IP) or intravenous (IV) injection of 100 μl of the same lupus serum as used in the intradermal injection. We did not find any histologic evidence of skin inflammation induced by intraperitoneal or intravenous injection of 100 μl of lupus serum (Fig. 1D). This result indicates that skin inflammation cannot be induced by the same dose of systemic administration of lupus serum as intradermal injection. To evaluate whether lupus serum induces skin inflammation in other mouse strains, we examined six different mouse strains, including C57BL/6, Swiss, BALB/c, C3H/HeN, C3H/HeJ and B17. We found that skin inflammation was induced by lupus serum in all of these strains (Fig. 1E). This result suggests that lupus serum can induce skin inflammation in various mouse strains, not in a specific mouse strain. 3.2. Relationship between skin inflammation and SLE activity To understand whether the severity of lupus serum-induced skin inflammation correlates with SLE disease activity, we performed the following experiments. First, we investigated the relationship between skin inflammation and SLE disease severity. To this aim, we treated mice with an intradermal injection of serum from patients whose
Fig. 2. Relationship between lupus serum-induced skin inflammation and disease activity. (A–B) Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of lupus patients with or without skin disease and SLEDAI N4 or SLEDAI ≤4. (C) Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of serum (100 μl) from lupusprone MRL/lpr mice with skin injury at 16 weeks of age and without skin injury at 10 weeks of age (100 μl). (D) Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of serum (100 μl) from lupus-prone MRL/lpr mice with proteinuria (+) and proteinuria (++++) at 20 weeks of age. (n = 5 per group) *p b 0.05; **p N 0.05.
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SLEDAI was ≤4 or N4. We found that the severity of skin inflammation was not significantly different between the two groups (Fig. 2A). Second, we compared the severity of skin inflammation induced by lupus serum with or without a skin manifestation. We found that the severity of skin inflammation was more severe in mice injected with serum from lupus patients with a skin manifestation than that of patients without a skin manifestation (Fig. 2B). Third, we compared the severity of skin inflammation induced by serum from MRL/lpr mice with or without a skin injury manifestation. We found that serum from MRL/lpr mice with skin injury induced more severe skin inflammation than serum from MRL/ lpr mice without skin injury (Fig. 2C). Finally, we compared the severity of skin inflammation induced by serum from MRL/lpr mice with mild proteinuria (+) or severe proteinuria (++++). We found no significant difference between the two groups (Fig. 2D). These data indicate that lupus serum-induced skin inflammation is closely related to the presence of skin disease activity but not to systemic disease severity. 3.3. The role of IgG, anti-dsDNA, anti-Ro Ab and C3/C4 in skin inflammation induced by lupus serum Because IgG is a major component of serum, we determined the role of IgG in the development of lupus serum-induced skin inflammation. To this aim, we used protein G agarose beads to remove IgG from lupus serum. Then, we injected lupus serum with IgG depletion and lupus serum without IgG depletion into the skin of normal mice. We found that the severity of skin inflammation was significantly decreased in mice injected with IgG-depleted lupus serum compared with mice injected with IgG-nondepleted lupus serum (Fig. 3A, left). We also collected IgG from lupus serum, injected it into the skin of mice and found that IgG from lupus serum can directly induce skin inflammation (Fig. 3A, right). This result indicates that IgG is a major contributor to the development of lupus serum-induced skin inflammation. Because anti-dsDNA antibody (Ab) has been used to indicate tissue damage, such as that in the kidney and brain [2,17], we wondered whether anti-dsDNA Ab is involved in the pathogenesis of skin inflammation induced by lupus serum. To this aim, we performed the following experiments. First, we injected intradermally the same dose of antidsDNA antibody-positive and –negative serum into the skin of normal mice. We found no significant difference in the severity of skin
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inflammation between the group of mice injected with anti-dsDNA antibody-positive serum and the group of mice injected with antidsDNA antibody-negative serum (Fig. 3B). Then, we injected the same dose of serum with a high titer and low titer of anti-dsDNA into the skin of normal mice. We found that the severity of skin inflammation was not different between the group of mice injected with a high titer of anti-dsDNA Ab and that with a low titer of anti-dsDNA Ab (Fig. 3C). The results demonstrate that the severity of skin inflammation induced by lupus serum was not related to the anti-dsDNA antibody. Because anti-Ro Ab is involved in the pathogenesis of skin injury in lupus [19,20], we investigated whether anti-Ro Ab is related to the skin inflammation induced by lupus serum. We injected anti-Ropositive serum and anti-Ro-negative serum into the skin of normal mice. A histopathological examination demonstrated no significant difference in the severity of skin inflammation between the group of mice injected with anti-Ro-positive serum and the group of mice injected with anti-Ro-negative serum (Fig. 3D). These data do not support that skin inflammation induced by lupus serum is related to anti-Ro Ab. Because complement (C) is involved in the pathogenesis of tissue damage in SLE [23], we determined whether complement is involved in skin inflammation induced by lupus serum. To this end, mice were treated with an intradermal injection of serum from lupus patients with high or low levels of C3/C4. We did not observe a significant difference in the severity of skin inflammation between the group of mice with high levels of C3/C4 and that with low levels of C3/C4 (Fig. 3E). These data suggest that low C3/C4 is not related to the skin inflammation induced by lupus serum. 3.4. Histopathological features of skin inflammation A histopathological examination showed keratinocyte hypertrophy and infiltrating cells in the epidermal, dermal and surrounding blood vessels in skin sections (Fig. 1A). To determine when skin inflammation appears, the peak of skin inflammation and the duration of skin inflammation, we investigated the kinetics of skin inflammation induced by one injection of lupus serum. We observed that skin inflammation appeared 3 h after intradermal injection and lasted for at least 14 days. The maximal severity of skin inflammation was noted on day 3 after intradermal injection of lupus serum (Fig. 4A). To assess whether skin
Fig. 3. The role of IgG, autoantibody and complement in lupus serum-induced skin inflammation. Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of 100 μl of lupus patient serum under the following conditions: with or without IgG depletion (A, left), lupus IgG (A, right); anti-dsDNA Ab-positive or -negative (B); high or low titer of anti-dsDNA Ab (C); anti-Ro Ab-positive or -negative (D); low or high titer of C3/C4 (E). (n = 5 per group) *p b 0.05; **p N 0.05.
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Fig. 4. Histopathological and immunohistochemical features of skin inflammation induced by lupus serum. (A) Kinetics of skin inflammation induced by serum from a lupus patient with skin disease. (B) The severity of skin inflammation induced by various volumes of lupus serum from a patient with skin disease 3 d after intradermal injection. (C) The severity of skin inflammation induced by a single intradermal injection and multiple intradermal injections. (D) Immunohistochemical staining of a skin lesion induced by lupus serum, showing the expression of CD11b, CD44, CD20 and CD3 in infiltrating cells (brown) 3 d after intradermal injection. *p b 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
inflammation is related to the volume of injected lupus serum, different doses of serum were intradermally injected into mice. We found that 5 μl is the lowest dose that could trigger skin inflammation, and the severity of skin inflammation depends on the volume of injected lupus serum (Fig. 4B). To study the effects of repeated exposure to lupus serum, we injected 50 μl of lupus serum intradermally on days 0, 7 and 14. On day 21, the mice were sacrificed, and their skins were analyzed by histological examination. The severity of skin inflammation was more severe with repetitive injections than with a single injection (Fig. 4C). 3.5. Immunohistochemical features of skin inflammation In skin lesions induced by lupus serum, there are a large proportion of mononuclear cells, which stained positive with CD11b. These stained cells, having the morphology of macrophages (i.e., mononuclear cells), were found within the dermis layer. The predominance of CD11b + cells and CD44+ cells was evident at all stages of inflammation. Few CD3+ T cells were found on days 3 and 7 but they constituted b5% of the total number of inflammatory cells. CD20+ B cells were not found in the skin inflammation throughout the experimental period (Fig. 4D). 3.6. Immunoglobulin and autoantibodies To understand whether intradermal injection of lupus serum affects total serum IgG levels in host mice, we investigated the levels of serum in mice with intradermal injection of lupus serum and the PBS control. The results demonstrated that total mouse serum IgG levels did not significantly increase in comparison with PBS-injected control mice within 3 weeks after serum injection (Fig. 5A). The results showed no difference in serum levels of IgG and IgM antibodies specific for dsDNA and ssDNA between lupus serum-injected mice and PBS-injected control mice (data not shown). 3.7. Cytokine levels in serum To understand whether intradermal injection of lupus serum affects cytokine levels in serum, we measured cytokine levels in serum from
mice with lupus serum-induced skin inflammation. We found no significant difference in serum IL-6 and TNF between mice with lupus serum injection and mice with PBS injection (Fig. 5B, C). These data indicate that local intradermal injection of lupus serum did not affect systemic cytokine levels. 3.8. Expression of cytokine, chemokine and NF-kB in skin lesion induced by lupus serum Because TNF is a proinflammatory cytokine and plays an important role in inflammatory skin diseases, TNF exerts its effect through its receptor; thus, we determined TNFR expression in skin inflammatory lesions induced by lupus serum using immunohistochemical staining. We found that TNFR1 is expressed more prominently than TNFR2 in lupus serum-induced skin lesions (Fig. 6A). This finding indicates that TNFR1 exerts an important role in the pathogenesis of lupus seruminduced skin inflammation. Because TNF/TNFR1 signaling mediates NF-kB activation and NF-kB is an intracellular molecule that regulates the production of proinflammatory cytokines [27], we investigated the expression of NF-kB in skin lesions induced by lupus serum. The result showed that p65NF-kB expression was markedly increased in inflammatory lesions caused by lupus serum compared with healthy serum (Fig. 6B). Monocytes migrate to inflammatory sites from blood vessels in response to increased expression of chemokines. MCP-1 was shown to attract monocytes to inflamed skin in MRL/lpr mice [28]. We evaluated MCP-1 expression in skin lesions induced by lupus serum using immunohistochemical staining. The result demonstrated that MCP-1 expression was largely increased in the skin at the site of intradermal injection of SLE serum compared with healthy serum in normal mice (Fig. 6C). 3.9. Synergism of TLR9 ligand and lupus serum in triggering skin inflammation Because keratinocytes express TLR9 in skin lesions of MRL/lpr mice and the TLR9 ligand mediates monocyte/macrophage activation and induces NF-kB activation and inflammation [27], lupus serum may contain anti-dsDNA antibody that can bind to the TLR9 ligand. Thus, we wondered whether CpG DNA and lupus serum act in synergy with
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Fig. 5. Levels of IgG, IL-6 and TNF in serum of mice at various time points after intradermal injection of lupus patient serum or PBS.
respect to the induction of skin inflammation. We evaluated mice that had been injected intradermally with suboptimal doses of each of the substances simultaneously. The results demonstrated that severe skin inflammation was observed in mice with intradermal injection of suboptimal doses of both CpG DNA and lupus serum compared with mice injected with each substance individually (Fig. 7A). To confirm the role of CpG DNA in skin inflammation induced by lupus serum, we further assessed whether an inhibitory oligonucleotide suppresses skin inflammation induced by lupus serum. We found that the severity of skin inflammation was decreased in mice with intradermal injection of lupus serum in the presence of inhibitory oligonucleotide compared with that in the absence of inhibitory oligonucleotide (Fig. 7B). These data indicate that TLR9 ligand and lupus serum act synergistically to trigger skin inflammation. 4. Discussion Our results demonstrate features of lupus serum-induced skin inflammation. Serum from SLE patients and lupus-prone mice induced skin inflammation that is not caused by LPS contamination. Histopathological signs of skin inflammation were evident within 3 h and lasted for at least two weeks. IgG was responsible for the induction of skin
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inflammation. Skin inflammation was characterized by an influx of monocytic, Mac-1+ cells and accumulated CD11C+ cells and by a scarcity of T lymphocytes. The disease was related to lupus patients with skin clinical manifestation but not to SLE disease activity and antidsDNA antibody. The disease is characterized locally by the expression of TNFR1, NF-kB and MCP-1 and not by increased levels of circulating IL-6, TNF and IgG. The skin inflammation is caused by lupus serum not by LPS contamination because there was not a significant difference in the severity of skin inflammation between the LPS-responding mouse strain and LPSresistant mouse strain. The possibility of traumatic/surgical injury can also be excluded because the injection of PBS or normal serum did not result in skin inflammation. The severity of skin inflammation depends on the dose of lupus serum locally on the skin. The skin inflammation triggered by 100 μl of lupus serum was more severe than the skin inflammation triggered by 50 or 25 μl of lupus serum. Skin inflammation only developed following local injection of lupus serum and not after systemic injection of lupus serum. Local injection compared to systemic injection should result in larger concentrations of lupus serum in the skin. Skin inflammation is related to pathological components containing serum. Compared to healthy individual serum, lupus serum contains a large amount of IgG and autoantibodies. Skin IgG deposition is one feature of the skin found in lupus patients. IgG deposition to the skin means that the skin inflammation is related to the dose of IgG. The same volume of lupus serum contains much more IgG than healthy serum; thus, the skin inflammation was induced by lupus serum not by healthy serum when the same volume of lupus serum and healthy serum were injected intradermally. Surprisingly, the skin inflammation triggered by lupus serum was not related to different types of autoantibodies, including anti-dsDNA and anti-Ro autoantibody, but was related to the skin disease that lupus patients suffer from. SLE patients have high levels of different autoantibodies in the serum. Anti-dsDNA antibodies, typically present in SLE sera, have been demonstrated to contribute to central nervous system [17] and kidney damage [2]. Although anti-dsDNA-positive human lupus sera caused skin injury, we cannot claim that anti-DNA Abs are directly involved in the expression of skin inflammation because the injection of monoclonal anti-DNA Abs and the injection of sera with progressively higher titers of anti-DNA antibodies failed to induce significant skin inflammation. Anti-Ro antibodies have been shown in skin lesions in lupus patients and animal models [4,29]. Anti-Ro antibodies bind to keratinocytes expressing the Ro antigen on the surface membrane, and it has been postulated that they facilitate antibodydependent cell cytotoxicity by infiltrating cytotoxic cells [4]. However, we found that the severity of skin inflammation induced by anti-Ro antibody-containing lupus serum was not significantly different between anti-Ro-negative- and anti-Ro-positive-containing lupus serum. It is possible that other autoantibodies, including anti-histone, antiphospholipid antibodies [2], anti-acidic ribosomal protein P0 (antiRPLP0), anti-galectin 3 antibodies or anti-Ro52 autoantibodies [18,19], may elicit skin inflammation [2,5]. SLE serum has been shown to contain anti-TCR/CD3 autoantibodies that can suppress interleukin-2 production [30]. Thus, our data indicate that we need to further investigate whether these autoantibodies are related to the skin inflammation induced by lupus serum. Our study demonstrates that there is synergism between the TLR9 ligand and lupus serum in mediating skin inflammation. This synergism may have two possibilities. CpG DNA directly stimulates dendritic cells to produce proinflammatory cytokines or may form immune complexes through binding to anti-DNA Ab in lupus serum. This result may explain how UV light enhances skin injury in lupus patients. Exposure to UV triggers keratinocyte necrosis in lupus patients and the release of nuclear antigen, including ds-DNA, which can directly activate dendritic cells to produce proinflammatory cytokines or may form immune complexes through binding to anti-dsDNA Ab deposited in the skin.
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Fig. 6. Expression of TNFR, MCP-1 and NF-kB in skin lesions induced by lupus serum. Immunohistochemical staining shows expression of TNFR1 and TNFR2 in skin lesions 3 d after intradermal inoculation of serum (100 μl) from a lupus patient in C57BL/6 mice. (A). Immunohistochemical staining shows NF-kB (B) and MCP-1 (C) expression in skin lesions 3 d after intradermal injection of serum from a lupus patient and a healthy control in C57BL/6 mice.
The local injection of lupus serum into the skin stimulated a local inflammatory response but did not affect the systemic response. In skin lesions induced by lupus serum, the expression of TNF, TNFR1, NF-kB, MCP-1 and iNOS was increased, but the levels of cytokines, such as TNF, IL-1 and IL-6, immunoglobulin and anti-dsDNA antibody in serum were not affected compared to controls. Although our study suggests that intradermal injection of small doses of lupus serum
cannot affect the systemic response, another study has demonstrated that UV light may promote local skin injury in lupus patients, mediate flares of systemic disease and affect kidney damage [31]. This finding suggests that skin injury may promote systemic pathological progression, such as nephritis flare. Thus, it is necessary to develop an effective prevention and treatment strategy for the skin injuries of lupus patients.
Fig. 7. Lupus serum and CpG DNA synergize in the induction of skin inflammation. (A) Severity of skin inflammation in C57BL/6 mice (n = 5 per group) injected intradermally with CpG ODN 1668 (5 μg), lupus serum (25 μl) or a combination of CpG ODN 1668 (5 μg) and lupus serum (25 μl). (B) Severity of skin inflammation in C57BL/6 mice injected intradermally with lupus serum (100 μl), inhibitory oligonucleotide (10 μg) or combination of inhibitory oligonucleotide (10 μg) and lupus serum (100 μl). After 3 d, the mice were sacrificed, and a histopathological examination was performed. Values are the mean and SD of the severity score. (n = 5 per group) *p b 0.05; **p N 0.05.
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5. Conclusion All data describing the feature of lupus serum-induced skin inflammation suggest that skin inflammation induced by lupus serum is a useful animal model to study the skin injury pathogenesis of SLE. This novel animal model will allow us to easily dissect the pathogenesis of lupus patients and promote the development of a therapeutic target against skin injuries in patients with SLE. Acknowledgments Financial support was provided by the Research Initiating Fund of Nanjing Medical University (KY101RC071203) and NIH 5R21 A1083762 (Deng GM). We thank Dr. George Tsokos at Beth Israel Deaconess Medical Center for his help and support. References [1] G.C. Tsokos, Systemic lupus erythematosus, N. Engl. J. Med. 365 (2011) 2110–2121. [2] A. Rahman, D.A. Isenberg, Systemic lupus erythematosus, N. Engl. J. Med. 358 (2008) 929–939. [3] R. Cervera, M.A. Khamashta, J. Font, G.D. Sebastiani, A. Gil, P. Lavilla, et al., The European Working Party on systemic lupus erythematosus. systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 1000 patients, Medicine (Baltimore) 72 (1993) 113–124. [4] H.J. Lee, A.A. Sinha, Cutaneous lupus erythematosus: understanding of clinical features, genetic basis, and pathobiology of disease guides therapeutic strategies, Autoimmunity 39 (2006) 433–444. [5] G.M. Deng, G.C. Tsokos, Pathogenesis and targeted treatment of skin injury of SLE, Nat. Rev. Rheumatol. 11 (2015) 663–669. [6] A. Kuhn, A. Landmann, The classification and diagnosis of cutaneous lupus erythematosus, J. Autoimmun. 48-49 (2014) 14–19. [7] E.D. Privette, V.P. Werth, Update on pathogenesis and treatment of CLE, Curr. Opin. Rheumatol. 25 (2013) 584–590. [8] L.G. Okon, V.P. Werth, Cutaneous lupus erythematosus: diagnosis and treatment, Best Pract. Res. Clin. Rheumatol 27 (2013) 391–404. [9] V. Oke, M. Wahren-Herlenius, Cutaneous lupus erythematosus: clinical aspects and molecular pathogenesis, J. Intern. Med. 273 (2013) 544–554. [10] M.A. Vera-Recabarren, M. García-Carrasco, M. Ramos-Casals, C. Herrero, Comparative analysis of subacute cutaneous lupus erythematosus and chronic cutaneous lupus erythematosus: clinical and immunological study of 270 patients, Br. J. Dermatol 162 (2010) 91–101. [11] A. Kuhn, D. Bein, G. Bonsmann, The 100th anniversary of lupus erythematosus tumidus, Autoimmun. Rev. 8 (2009) 441–448. [12] T.T. Provost, Lupus band test, Int. J. Dermatol. 20 (1981) 475–481. [13] M.V. Dahl, Usefulness of direct immunofluorescence in patients with lupus erythematosus, Arch. Dermatol. 119 (1983) 1010–1017.
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[14] Q. Yin, X. Xu, Y. Lin, J. Lv, L. Zhao, He R, Ultraviolet B irradiation induces skin accumulation of plasmacytoid dendritic cells: a possible role for chemerin, Autoimmunity 47 (2014) 185–192. [15] K.A. Kirou, E. Gkrouzman, Anti-interferon alpha treatment in SLE, Clin. Immunol. 148 (2013) 303–312. [16] E. Reefman, H. Kuiper, P.C. Limburg, C.G. Kallenberg, M. Bijl, Type I interferons are involved in the development of ultraviolet B-induced inflammatory skin lesions in systemic lupus erythematosus patients, Ann. Rheum. Dis. 67 (2008) 11–18. [17] L.A. DeGiorgio, K.N. Konstantinov, S.C. Lee, J.A. Hardin, B.T. Volpe, B. Diamond, A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus, Nat. Med. 7 (2001) 1189–1193. [18] Z.R. Shi, C.X. Cao, G.Z. Tan, L. Wang, Association of anti-acidic ribosomal protein p0 and anti-galectin 3 antibodies with the development of skin lesions in systemic lupus erythematosus, Arthritis Rheumatol. 67 (2015) 193–203. [19] V. Oke, I. Vassilaki, A. Espinosa, L. Strandberg, V.K. Kuchroo, F. Nyberg, et al., High Ro52 expression in spontaneous and UV-induced cutaneous inflammation, J. Investig. Dermatol. 129 (2009) 2000–2010. [20] A. Espinosa, V. Dardalhon, S. Brauner, A. Ambrosi, R. Higgs, F.J. Quintana, et al., Loss of the lupus autoantigen Ro52/Trim21 induces tissue inflammation and systemic autoimmunity by disregulating the IL-23-Th17 pathway, J. Exp. Med. 206 (2009) 1661–1671. [21] F. Furukawa, H. Tanaka, K. Sekita, T. Nakamura, Y. Horiguchi, Y. Hamashima, Dermatopathological studies on skin lesions of MRL mice, Arch. Dermatol. Res. 276 (1984) 186–194. [22] G.M. Deng, G.C. Tsokos, Cholera toxin B accelerates disease progression in lupusprone mice by promoting lipid raft aggregation, J. Immunol. 181 (2008) 4019–4026. [23] G.M. Deng, L. Liu, V.C. Kyttaris, G.C. Tsokos, Lupus serum IgG induces skin inflammation through the TNFR1 signaling pathway, J. Immunol. 184 (2010) 7154–7161. [24] G.M. Deng, L. Liu, G.C. Tsokos, Targeted tumor necrosis factor receptor I preligand assembly domain improves skin lesions in MRL/lpr mice, Arthritis Rheum. 62 (2010) 2424–2431. [25] G.M. Deng, L. Liu, F.R. Bahjat, P.R. Pine, G.C. Tsokos, Suppression of skin and kidney disease by inhibition of spleen tyrosine kinase in lupus-prone mice, Arthritis Rheum. 62 (2010) 2086–2092. [26] G.M. Deng, M. Nilsson, M. Verdrengh, L.V. Collins, A. Tarkowski, Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis, Nat. Med. 5 (1999) 702–705. [27] G.M. Deng, M. Verdrengh, Z.Q. Liu, A. Tarkowski, The major role of macrophages and their product tumor necrosis factor alpha in the induction of arthritis triggered by bacterial DNA containing CpG motifs, Arthritis Rheum. 43 (2000) 2283–2289. [28] G.H. Tesch, S. Maifert, A. Schwarting, B.J. Rollins, V.R. Kelley, Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice, J. Exp. Med. 190 (1999) 1813–1824. [29] L.A. Lee, K.K. Gaither, S.N. Coulter, D.A. Norris, J.B. Harley, Pattern of cutaneous immunoglobulin G deposition in subacute cutaneous lupus erythematosus is reproduced by infusing purified anti-Ro (SSA) autoantibodies into human skingrafted mice, J. Clin. Invest. 83 (1989) 1556–1562. [30] Y.T. Juang, Y. Wang, E.E. Solomou, Y. Li, C. Mawrin, K. Tenbrock, et al., Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV, J. Clin. Invest. 115 (2005) 996–1005. [31] K.L. Clark, T.J. Reed, S.J. Wolf, L. Lowe, J.B. Hodgin, J.M. Kahlenberg, Epidermal injury promotes nephritis flare in lupus-prone mice, J. Autoimmun. (2015) (pii: S08968411(15)30023–8).
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Clinical Immunology journal homepage: www.elsevier.com/locate/yclim
The features of skin inflammation induced by lupus serum Lena Liu b, Guangqion Xu a, Hui Dou a, Guo-Min Deng a,b,c,⁎ a b c
Key Lab of Antibody Techniques of Ministry of Health, Nanjing Medical University, Nanjing, China Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
a r t i c l e
i n f o
Article history: Received 16 February 2016 accepted with revision 17 February 2016 Available online 18 February 2016 Keywords: Systemic lupus erythematosus Lupus serum IgG Skin inflammation
a b s t r a c t We recently developed a model of lupus serum-induced skin inflammation, which was used to study the pathogenesis of skin injury in systemic lupus erythematosus (SLE). We further characterized the features of lupus serum-induced skin inflammation. This skin inflammation was evident within 3 h and lasted for at least two weeks. The skin inflammation was characterized by an influx of monocytic, CD11b + cells and by a scarcity of T and B lymphocytes. Depletion of IgG from the serum abrogated the skin inflammatory response. The skin inflammation was related to lupus patients' skin history but not to SLE disease activity and type of autoantibody. The expression of TNFR1, NF-kB and MCP-1 was increased locally in skin lesions. The TLR9 ligand and lupus serum act synergistically to trigger skin inflammation. These findings suggest that this novel model is valuable for the study of the pathogenesis and therapy of skin injury in SLE. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by high levels of autoantibody and multi-organ tissue damage, including kidney and skin damage [1,2]. Skin injury is second most common clinical manifestation in patients with SLE [3,4]. The molecular and cellular mechanisms involved in the expression of cutaneous LE remain unclear [5]. The skin injury of SLE can be divided into lupus erythematous (LE)specific or LE-nonspecific manifestations by histologic analysis of biopsy specimens [6]. The LE-nonspecific manifestations are commonly associated with systemic organ manifestations or other autoimmune diseases [6]. The LE-specific cutaneous manifestations may present without or with less severe systemic organ involvement and are further classified into several subtypes, including acute (A) CLE, subacute (S) CLE, chronic (C) CLE and intermittent (I) CLE, which was proposed to be a separate category for LE tumidus in 2004 and ICLE is not universally accepted [7–9]. ACLE, SCLE and CCLE are three common clinical subtypes of cutaneous LE. Localized ACLE is often referred to as the ‘malar rash’ or ‘butterfly rash’ of SLE, whereas generalized ACLE is frequently referred to as the ‘SLE rash’ [9]. The skin injuries share the following features of a lichenoid tissue reaction: hyperkeratosis; epidermal atrophy; liquefactive degeneration of the epidermal basal-cell layer; mononuclear cell infiltrates focused at the dermo-epidermal junction, perivascular areas and perifollicular areas; thickening of the basal membrane; and melanin pigment incontinence. ACLE typically presents abruptly in the context of a systemic disease, and almost all patients ⁎ Corresponding author at: Nanjing Medical University, China. E-mail address: [email protected] (G.-M. Deng).
http://dx.doi.org/10.1016/j.clim.2016.02.007 1521-6616/© 2016 Elsevier Inc. All rights reserved.
develop SLE [5]. ICLE seems to be a purely dermatological disease [10, 11]. The deposition of immune complexes containing IgG, IgM and complement (C) 3 is typically observed at the dermo-epidermal junction and is defined as a positive ‘lupus band’ test [12,13]. The production of autoantibodies directed against nuclear antigens and a myriad of other autoantigens characterizes SLE. In the skin, the invariably observed keratinocyte apoptosis may result in the cell surface expression and release of self-antigens, including DNA, Ro/SSA, La/SSB and histones [2,5]. Although exposure to UV and other environmental triggers may contribute to the initiation of cutaneous LE by triggering the apoptosis of keratinocytes, it is still unclear how the inflammatory process begins and is sustained [2,5]. IFN-α plays an important role in the pathogenesis of lupus erythematosus skin lesions [14]. Large numbers of IFN-producing plasmacytoid dendritic cells are also found in SLE skin lesions [15,16]. Tissue damage in SLE is associated with autoantibody production and immune complex formation and deposition [2]. An anti-doublestranded DNA antibody has been linked to kidney [2] and brain pathology [17]. Anti-acidic ribosomal protein P0 (anti-RPLP0) and antigalectin 3 antibodies are related to the development of skin lesions in SLE [18]. Ro52 is a common target of circulating autoantibodies in SLE. Ro52 is highly expressed in spontaneous and UV-induced cutaneous inflammation in CLE; approximately 80% of cells within infiltrates of CLE lesions are positively stained for Ro52 in the dermis [19]. The loss of autoantigen Ro52 induces skin inflammation and systemic autoimmunity [20]. This result indicates that Ro52 exerts an important regulatory role in lupus skin lesions. Apparently, Autoantigens bind to keratinocytes undergoing apoptosis and contribute to the inflammatory process [2]. MRL/lpr mice are a common animal model of SLE and spontaneously develop lupus-like clinical manifestations characterized by
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high levels of autoantibodies and multiorgan tissue damage, including skin injury [21,22]. These observations indicate that autoantibodies may play an important role in the expression of cutaneous lesions in SLE and led us to hypothesize that lupus serum induces skin inflammation. Recently, we have confirmed the hypothesis that serum from lupus patients and lupus-prone mice induced skin inflammation [23]. We also found that an animal model of lupus serum-induced skin inflammation is a valuable tool to investigate the pathogenesis and therapy of skin injuries in SLE [24,25]. In this paper, we further defined the features of skin inflammation induced by lupus serum.
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2.3. Histopathological examination Histopathological examination of skin was performed after routine fixation and paraffin embedding of the tissue. Tissue sections from the skin were cut and stained with hematoxylin and eosin. All slides were coded and evaluated in a ‘blinded to sample’ identity manner. The severity of skin inflammation was scored 0–4 as follows: grade 0, normal; grade 1, hyperplasia of epidermis; and grades 2–4, different amounts of infiltrating inflammatory cells in the skin with or without hyperplasia of epidermis [24]. 2.4. Immunohistochemical examination
2. Materials and methods 2.1. Mice and sera C57BL/6, BALB/c, Swiss and MRL/lpr mice were purchased from JAX Labs (Cold Harbor, USA). All mice were housed in the animal facility of the Beth Israel Deaconess Medical Center and Nanjing Medical University. Sera were collected from patients with SLE and MRL/lpr mice at different ages and normal C57BL/6 mice. Animal and human use protocols were approved by appropriate Beth Israel Deaconess Medical Center committees and by Nanjing Medical University committees. Sera were collected from patients with SLE and controls from Beth Israel Deaconess Medical Center and Jiangsu Province Hospital. Eighteen patients fulfilling ≥4 of 11 revised criteria of the American College of Rheumatology for the classification of SLE were studied. All patients had SLE disease-activity index (SLEDAI) scores ranging from 0 to 12. Medications were discontinued ≥24 h before venipuncture. Ten normal women served as controls in this study. Sera were collected from MRL/ lpr mice of different ages and normal C57BL/6 mice.
2.2. Injection protocol procedures Different doses of lupus serum were injected intradermally in the back of the neck of mice of different strains. All of the intradermal injections were performed on anesthetized mice [24].
After deparaffinization and antigen retrieval, samples were stained with antibodies to CD4 + (GK1.5), CD8 + (53.6.7), NF-κB and MCP-1 (Santa Cruz, CA) followed by incubation with biotinylated secondary antibodies, avidin-biotin-peroxidase complexes and 3-amino-9-ethylcarbazole containing H2O2. All sections were counterstained with Mayer's hematoxylin [24]. 2.5. Immunoglobulins Serum levels of IgG were measured by an enzyme-linked immunosorbent assay (ELISA). Antisera and immunoglobulin standards specific for IgG were purchased from Sigma. 2.6. Autoantibodies Serum antibody levels to denatured or single-stranded DNA (ssDNA) and to native or double-stranded DNA (dsDNA), were measured by ELISA using methylated bovine serum albumin (10 μg/ml) to precoat the wells, followed by coating with 50 μg of heat-denatured (boiled for 20 m and then cooled rapidly on ice) calf thymus DNA (Sigma) per milliliter or native calf thymus DNA, as previously described [23]. 2.7. Cytokine levels The cytokine level in mouse serum was determined by ELISA.
Fig. 1. Lupus serum induces skin inflammation. (A) Representative histopathological photomicrograph. Severity of skin inflammation of C57BL/6 mice sacrificed 3 d after intradermal inoculation of serum (100 μl) from a lupus patient with skin disease, a healthy control and PBS. (B) The severity of skin inflammation of LPS-responding C3H/HeN and LPSnonresponding C3H/HeJ mice 3 d after intradermal inoculation of lupus serum (100 μl) (n = 5 per group). (C) The severity of skin inflammation in C57BL/6 mice (n = 5 per group) with intradermal injection of 100 μl of serum from MRL/lpr mice and C57BL/6 mice at 16 weeks of age. (D) The severity of skin inflammation in C57BL/6 mice (n = 5 per group) with intradermal injection (ID), intra-venous injection or intraperitoneal injection of 100 μl of lupus serum. (E) Incidence of skin inflammation in various mouse strains with intradermal injection of 100 μl of serum from lupus patients. *p b 0.05; **p N 0.05.
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2.8. Statistical analysis Statistical evaluations were analyzed using Student's t-test. p ≤ 0.05 was considered statistically significant.
3. Results 3.1. Induction of skin inflammation by lupus serum Because IgG deposited in the skin of patients with SLE and lupusprone mice comes from blood circulation, we hypothesized that lupus serum containing IgG might induce skin inflammation. We injected PBS and serum from patients with SLE and healthy individuals directly into murine skin. Histopathological examination demonstrated that skin inflammation developed in mice with intradermal injection of serum from patients with SLE but not from healthy individuals and PBS (Fig. 1A). This result indicates that lupus serum can induce skin inflammation and that the skin inflammation was not caused by the surgical procedure because the skin inflammation did not develop in mice with intradermal injection of healthy control serum and PBS. Because skin inflammation induced by lupus serum might be caused by LPS contamination in lupus serum, we performed further investigations to exclude this possibility. To this aim, we injected lupus serum into the skin of LPS-nonresponder C3H/HeJ mice and LPS-resistant C3H/HeJ mice [26]. The results demonstrated that there was no difference in the severity and incidence of skin inflammation upon injection of lupus serum into LPS-resistant strain C3H/HeJ mice or their congenic LPS-responder strain C3H/HeN mice (Fig. 1B); in contrast, intradermal injection of LPS induced skin inflammation in C3H/HeN mice but not in C3H/HeJ mice (Fig. 1B). This result indicates that the induction of
skin inflammation was due to lupus serum rather than LPS contamination. To further define that skin inflammation is caused by lupus serum, we determined whether serum from lupus-prone mice induces skin inflammation. To this aim, we intradermally (ID) injected serum from lupus MRL/lpr mice and C57BL/6 mice at 16 weeks of age into the skin of normal C57BL/6 mice. We found that skin inflammation developed in mice injected with serum from lupus MRL/lpr mice but not from C57BL/6 mice (Fig. 1C). To further assess whether systemic administration of lupus serum might induce skin inflammation, we treated mice with intraperitoneal (IP) or intravenous (IV) injection of 100 μl of the same lupus serum as used in the intradermal injection. We did not find any histologic evidence of skin inflammation induced by intraperitoneal or intravenous injection of 100 μl of lupus serum (Fig. 1D). This result indicates that skin inflammation cannot be induced by the same dose of systemic administration of lupus serum as intradermal injection. To evaluate whether lupus serum induces skin inflammation in other mouse strains, we examined six different mouse strains, including C57BL/6, Swiss, BALB/c, C3H/HeN, C3H/HeJ and B17. We found that skin inflammation was induced by lupus serum in all of these strains (Fig. 1E). This result suggests that lupus serum can induce skin inflammation in various mouse strains, not in a specific mouse strain. 3.2. Relationship between skin inflammation and SLE activity To understand whether the severity of lupus serum-induced skin inflammation correlates with SLE disease activity, we performed the following experiments. First, we investigated the relationship between skin inflammation and SLE disease severity. To this aim, we treated mice with an intradermal injection of serum from patients whose
Fig. 2. Relationship between lupus serum-induced skin inflammation and disease activity. (A–B) Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of lupus patients with or without skin disease and SLEDAI N4 or SLEDAI ≤4. (C) Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of serum (100 μl) from lupusprone MRL/lpr mice with skin injury at 16 weeks of age and without skin injury at 10 weeks of age (100 μl). (D) Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of serum (100 μl) from lupus-prone MRL/lpr mice with proteinuria (+) and proteinuria (++++) at 20 weeks of age. (n = 5 per group) *p b 0.05; **p N 0.05.
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SLEDAI was ≤4 or N4. We found that the severity of skin inflammation was not significantly different between the two groups (Fig. 2A). Second, we compared the severity of skin inflammation induced by lupus serum with or without a skin manifestation. We found that the severity of skin inflammation was more severe in mice injected with serum from lupus patients with a skin manifestation than that of patients without a skin manifestation (Fig. 2B). Third, we compared the severity of skin inflammation induced by serum from MRL/lpr mice with or without a skin injury manifestation. We found that serum from MRL/lpr mice with skin injury induced more severe skin inflammation than serum from MRL/ lpr mice without skin injury (Fig. 2C). Finally, we compared the severity of skin inflammation induced by serum from MRL/lpr mice with mild proteinuria (+) or severe proteinuria (++++). We found no significant difference between the two groups (Fig. 2D). These data indicate that lupus serum-induced skin inflammation is closely related to the presence of skin disease activity but not to systemic disease severity. 3.3. The role of IgG, anti-dsDNA, anti-Ro Ab and C3/C4 in skin inflammation induced by lupus serum Because IgG is a major component of serum, we determined the role of IgG in the development of lupus serum-induced skin inflammation. To this aim, we used protein G agarose beads to remove IgG from lupus serum. Then, we injected lupus serum with IgG depletion and lupus serum without IgG depletion into the skin of normal mice. We found that the severity of skin inflammation was significantly decreased in mice injected with IgG-depleted lupus serum compared with mice injected with IgG-nondepleted lupus serum (Fig. 3A, left). We also collected IgG from lupus serum, injected it into the skin of mice and found that IgG from lupus serum can directly induce skin inflammation (Fig. 3A, right). This result indicates that IgG is a major contributor to the development of lupus serum-induced skin inflammation. Because anti-dsDNA antibody (Ab) has been used to indicate tissue damage, such as that in the kidney and brain [2,17], we wondered whether anti-dsDNA Ab is involved in the pathogenesis of skin inflammation induced by lupus serum. To this aim, we performed the following experiments. First, we injected intradermally the same dose of antidsDNA antibody-positive and –negative serum into the skin of normal mice. We found no significant difference in the severity of skin
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inflammation between the group of mice injected with anti-dsDNA antibody-positive serum and the group of mice injected with antidsDNA antibody-negative serum (Fig. 3B). Then, we injected the same dose of serum with a high titer and low titer of anti-dsDNA into the skin of normal mice. We found that the severity of skin inflammation was not different between the group of mice injected with a high titer of anti-dsDNA Ab and that with a low titer of anti-dsDNA Ab (Fig. 3C). The results demonstrate that the severity of skin inflammation induced by lupus serum was not related to the anti-dsDNA antibody. Because anti-Ro Ab is involved in the pathogenesis of skin injury in lupus [19,20], we investigated whether anti-Ro Ab is related to the skin inflammation induced by lupus serum. We injected anti-Ropositive serum and anti-Ro-negative serum into the skin of normal mice. A histopathological examination demonstrated no significant difference in the severity of skin inflammation between the group of mice injected with anti-Ro-positive serum and the group of mice injected with anti-Ro-negative serum (Fig. 3D). These data do not support that skin inflammation induced by lupus serum is related to anti-Ro Ab. Because complement (C) is involved in the pathogenesis of tissue damage in SLE [23], we determined whether complement is involved in skin inflammation induced by lupus serum. To this end, mice were treated with an intradermal injection of serum from lupus patients with high or low levels of C3/C4. We did not observe a significant difference in the severity of skin inflammation between the group of mice with high levels of C3/C4 and that with low levels of C3/C4 (Fig. 3E). These data suggest that low C3/C4 is not related to the skin inflammation induced by lupus serum. 3.4. Histopathological features of skin inflammation A histopathological examination showed keratinocyte hypertrophy and infiltrating cells in the epidermal, dermal and surrounding blood vessels in skin sections (Fig. 1A). To determine when skin inflammation appears, the peak of skin inflammation and the duration of skin inflammation, we investigated the kinetics of skin inflammation induced by one injection of lupus serum. We observed that skin inflammation appeared 3 h after intradermal injection and lasted for at least 14 days. The maximal severity of skin inflammation was noted on day 3 after intradermal injection of lupus serum (Fig. 4A). To assess whether skin
Fig. 3. The role of IgG, autoantibody and complement in lupus serum-induced skin inflammation. Severity of skin inflammation in C57BL/6 mice 3 d after intradermal injection of 100 μl of lupus patient serum under the following conditions: with or without IgG depletion (A, left), lupus IgG (A, right); anti-dsDNA Ab-positive or -negative (B); high or low titer of anti-dsDNA Ab (C); anti-Ro Ab-positive or -negative (D); low or high titer of C3/C4 (E). (n = 5 per group) *p b 0.05; **p N 0.05.
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Fig. 4. Histopathological and immunohistochemical features of skin inflammation induced by lupus serum. (A) Kinetics of skin inflammation induced by serum from a lupus patient with skin disease. (B) The severity of skin inflammation induced by various volumes of lupus serum from a patient with skin disease 3 d after intradermal injection. (C) The severity of skin inflammation induced by a single intradermal injection and multiple intradermal injections. (D) Immunohistochemical staining of a skin lesion induced by lupus serum, showing the expression of CD11b, CD44, CD20 and CD3 in infiltrating cells (brown) 3 d after intradermal injection. *p b 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
inflammation is related to the volume of injected lupus serum, different doses of serum were intradermally injected into mice. We found that 5 μl is the lowest dose that could trigger skin inflammation, and the severity of skin inflammation depends on the volume of injected lupus serum (Fig. 4B). To study the effects of repeated exposure to lupus serum, we injected 50 μl of lupus serum intradermally on days 0, 7 and 14. On day 21, the mice were sacrificed, and their skins were analyzed by histological examination. The severity of skin inflammation was more severe with repetitive injections than with a single injection (Fig. 4C). 3.5. Immunohistochemical features of skin inflammation In skin lesions induced by lupus serum, there are a large proportion of mononuclear cells, which stained positive with CD11b. These stained cells, having the morphology of macrophages (i.e., mononuclear cells), were found within the dermis layer. The predominance of CD11b + cells and CD44+ cells was evident at all stages of inflammation. Few CD3+ T cells were found on days 3 and 7 but they constituted b5% of the total number of inflammatory cells. CD20+ B cells were not found in the skin inflammation throughout the experimental period (Fig. 4D). 3.6. Immunoglobulin and autoantibodies To understand whether intradermal injection of lupus serum affects total serum IgG levels in host mice, we investigated the levels of serum in mice with intradermal injection of lupus serum and the PBS control. The results demonstrated that total mouse serum IgG levels did not significantly increase in comparison with PBS-injected control mice within 3 weeks after serum injection (Fig. 5A). The results showed no difference in serum levels of IgG and IgM antibodies specific for dsDNA and ssDNA between lupus serum-injected mice and PBS-injected control mice (data not shown). 3.7. Cytokine levels in serum To understand whether intradermal injection of lupus serum affects cytokine levels in serum, we measured cytokine levels in serum from
mice with lupus serum-induced skin inflammation. We found no significant difference in serum IL-6 and TNF between mice with lupus serum injection and mice with PBS injection (Fig. 5B, C). These data indicate that local intradermal injection of lupus serum did not affect systemic cytokine levels. 3.8. Expression of cytokine, chemokine and NF-kB in skin lesion induced by lupus serum Because TNF is a proinflammatory cytokine and plays an important role in inflammatory skin diseases, TNF exerts its effect through its receptor; thus, we determined TNFR expression in skin inflammatory lesions induced by lupus serum using immunohistochemical staining. We found that TNFR1 is expressed more prominently than TNFR2 in lupus serum-induced skin lesions (Fig. 6A). This finding indicates that TNFR1 exerts an important role in the pathogenesis of lupus seruminduced skin inflammation. Because TNF/TNFR1 signaling mediates NF-kB activation and NF-kB is an intracellular molecule that regulates the production of proinflammatory cytokines [27], we investigated the expression of NF-kB in skin lesions induced by lupus serum. The result showed that p65NF-kB expression was markedly increased in inflammatory lesions caused by lupus serum compared with healthy serum (Fig. 6B). Monocytes migrate to inflammatory sites from blood vessels in response to increased expression of chemokines. MCP-1 was shown to attract monocytes to inflamed skin in MRL/lpr mice [28]. We evaluated MCP-1 expression in skin lesions induced by lupus serum using immunohistochemical staining. The result demonstrated that MCP-1 expression was largely increased in the skin at the site of intradermal injection of SLE serum compared with healthy serum in normal mice (Fig. 6C). 3.9. Synergism of TLR9 ligand and lupus serum in triggering skin inflammation Because keratinocytes express TLR9 in skin lesions of MRL/lpr mice and the TLR9 ligand mediates monocyte/macrophage activation and induces NF-kB activation and inflammation [27], lupus serum may contain anti-dsDNA antibody that can bind to the TLR9 ligand. Thus, we wondered whether CpG DNA and lupus serum act in synergy with
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Fig. 5. Levels of IgG, IL-6 and TNF in serum of mice at various time points after intradermal injection of lupus patient serum or PBS.
respect to the induction of skin inflammation. We evaluated mice that had been injected intradermally with suboptimal doses of each of the substances simultaneously. The results demonstrated that severe skin inflammation was observed in mice with intradermal injection of suboptimal doses of both CpG DNA and lupus serum compared with mice injected with each substance individually (Fig. 7A). To confirm the role of CpG DNA in skin inflammation induced by lupus serum, we further assessed whether an inhibitory oligonucleotide suppresses skin inflammation induced by lupus serum. We found that the severity of skin inflammation was decreased in mice with intradermal injection of lupus serum in the presence of inhibitory oligonucleotide compared with that in the absence of inhibitory oligonucleotide (Fig. 7B). These data indicate that TLR9 ligand and lupus serum act synergistically to trigger skin inflammation. 4. Discussion Our results demonstrate features of lupus serum-induced skin inflammation. Serum from SLE patients and lupus-prone mice induced skin inflammation that is not caused by LPS contamination. Histopathological signs of skin inflammation were evident within 3 h and lasted for at least two weeks. IgG was responsible for the induction of skin
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inflammation. Skin inflammation was characterized by an influx of monocytic, Mac-1+ cells and accumulated CD11C+ cells and by a scarcity of T lymphocytes. The disease was related to lupus patients with skin clinical manifestation but not to SLE disease activity and antidsDNA antibody. The disease is characterized locally by the expression of TNFR1, NF-kB and MCP-1 and not by increased levels of circulating IL-6, TNF and IgG. The skin inflammation is caused by lupus serum not by LPS contamination because there was not a significant difference in the severity of skin inflammation between the LPS-responding mouse strain and LPSresistant mouse strain. The possibility of traumatic/surgical injury can also be excluded because the injection of PBS or normal serum did not result in skin inflammation. The severity of skin inflammation depends on the dose of lupus serum locally on the skin. The skin inflammation triggered by 100 μl of lupus serum was more severe than the skin inflammation triggered by 50 or 25 μl of lupus serum. Skin inflammation only developed following local injection of lupus serum and not after systemic injection of lupus serum. Local injection compared to systemic injection should result in larger concentrations of lupus serum in the skin. Skin inflammation is related to pathological components containing serum. Compared to healthy individual serum, lupus serum contains a large amount of IgG and autoantibodies. Skin IgG deposition is one feature of the skin found in lupus patients. IgG deposition to the skin means that the skin inflammation is related to the dose of IgG. The same volume of lupus serum contains much more IgG than healthy serum; thus, the skin inflammation was induced by lupus serum not by healthy serum when the same volume of lupus serum and healthy serum were injected intradermally. Surprisingly, the skin inflammation triggered by lupus serum was not related to different types of autoantibodies, including anti-dsDNA and anti-Ro autoantibody, but was related to the skin disease that lupus patients suffer from. SLE patients have high levels of different autoantibodies in the serum. Anti-dsDNA antibodies, typically present in SLE sera, have been demonstrated to contribute to central nervous system [17] and kidney damage [2]. Although anti-dsDNA-positive human lupus sera caused skin injury, we cannot claim that anti-DNA Abs are directly involved in the expression of skin inflammation because the injection of monoclonal anti-DNA Abs and the injection of sera with progressively higher titers of anti-DNA antibodies failed to induce significant skin inflammation. Anti-Ro antibodies have been shown in skin lesions in lupus patients and animal models [4,29]. Anti-Ro antibodies bind to keratinocytes expressing the Ro antigen on the surface membrane, and it has been postulated that they facilitate antibodydependent cell cytotoxicity by infiltrating cytotoxic cells [4]. However, we found that the severity of skin inflammation induced by anti-Ro antibody-containing lupus serum was not significantly different between anti-Ro-negative- and anti-Ro-positive-containing lupus serum. It is possible that other autoantibodies, including anti-histone, antiphospholipid antibodies [2], anti-acidic ribosomal protein P0 (antiRPLP0), anti-galectin 3 antibodies or anti-Ro52 autoantibodies [18,19], may elicit skin inflammation [2,5]. SLE serum has been shown to contain anti-TCR/CD3 autoantibodies that can suppress interleukin-2 production [30]. Thus, our data indicate that we need to further investigate whether these autoantibodies are related to the skin inflammation induced by lupus serum. Our study demonstrates that there is synergism between the TLR9 ligand and lupus serum in mediating skin inflammation. This synergism may have two possibilities. CpG DNA directly stimulates dendritic cells to produce proinflammatory cytokines or may form immune complexes through binding to anti-DNA Ab in lupus serum. This result may explain how UV light enhances skin injury in lupus patients. Exposure to UV triggers keratinocyte necrosis in lupus patients and the release of nuclear antigen, including ds-DNA, which can directly activate dendritic cells to produce proinflammatory cytokines or may form immune complexes through binding to anti-dsDNA Ab deposited in the skin.
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Fig. 6. Expression of TNFR, MCP-1 and NF-kB in skin lesions induced by lupus serum. Immunohistochemical staining shows expression of TNFR1 and TNFR2 in skin lesions 3 d after intradermal inoculation of serum (100 μl) from a lupus patient in C57BL/6 mice. (A). Immunohistochemical staining shows NF-kB (B) and MCP-1 (C) expression in skin lesions 3 d after intradermal injection of serum from a lupus patient and a healthy control in C57BL/6 mice.
The local injection of lupus serum into the skin stimulated a local inflammatory response but did not affect the systemic response. In skin lesions induced by lupus serum, the expression of TNF, TNFR1, NF-kB, MCP-1 and iNOS was increased, but the levels of cytokines, such as TNF, IL-1 and IL-6, immunoglobulin and anti-dsDNA antibody in serum were not affected compared to controls. Although our study suggests that intradermal injection of small doses of lupus serum
cannot affect the systemic response, another study has demonstrated that UV light may promote local skin injury in lupus patients, mediate flares of systemic disease and affect kidney damage [31]. This finding suggests that skin injury may promote systemic pathological progression, such as nephritis flare. Thus, it is necessary to develop an effective prevention and treatment strategy for the skin injuries of lupus patients.
Fig. 7. Lupus serum and CpG DNA synergize in the induction of skin inflammation. (A) Severity of skin inflammation in C57BL/6 mice (n = 5 per group) injected intradermally with CpG ODN 1668 (5 μg), lupus serum (25 μl) or a combination of CpG ODN 1668 (5 μg) and lupus serum (25 μl). (B) Severity of skin inflammation in C57BL/6 mice injected intradermally with lupus serum (100 μl), inhibitory oligonucleotide (10 μg) or combination of inhibitory oligonucleotide (10 μg) and lupus serum (100 μl). After 3 d, the mice were sacrificed, and a histopathological examination was performed. Values are the mean and SD of the severity score. (n = 5 per group) *p b 0.05; **p N 0.05.
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5. Conclusion All data describing the feature of lupus serum-induced skin inflammation suggest that skin inflammation induced by lupus serum is a useful animal model to study the skin injury pathogenesis of SLE. This novel animal model will allow us to easily dissect the pathogenesis of lupus patients and promote the development of a therapeutic target against skin injuries in patients with SLE. Acknowledgments Financial support was provided by the Research Initiating Fund of Nanjing Medical University (KY101RC071203) and NIH 5R21 A1083762 (Deng GM). We thank Dr. George Tsokos at Beth Israel Deaconess Medical Center for his help and support. References [1] G.C. Tsokos, Systemic lupus erythematosus, N. Engl. J. Med. 365 (2011) 2110–2121. [2] A. Rahman, D.A. Isenberg, Systemic lupus erythematosus, N. Engl. J. Med. 358 (2008) 929–939. [3] R. Cervera, M.A. Khamashta, J. Font, G.D. Sebastiani, A. Gil, P. Lavilla, et al., The European Working Party on systemic lupus erythematosus. systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 1000 patients, Medicine (Baltimore) 72 (1993) 113–124. [4] H.J. Lee, A.A. Sinha, Cutaneous lupus erythematosus: understanding of clinical features, genetic basis, and pathobiology of disease guides therapeutic strategies, Autoimmunity 39 (2006) 433–444. [5] G.M. Deng, G.C. Tsokos, Pathogenesis and targeted treatment of skin injury of SLE, Nat. Rev. Rheumatol. 11 (2015) 663–669. [6] A. Kuhn, A. Landmann, The classification and diagnosis of cutaneous lupus erythematosus, J. Autoimmun. 48-49 (2014) 14–19. [7] E.D. Privette, V.P. Werth, Update on pathogenesis and treatment of CLE, Curr. Opin. Rheumatol. 25 (2013) 584–590. [8] L.G. Okon, V.P. Werth, Cutaneous lupus erythematosus: diagnosis and treatment, Best Pract. Res. Clin. Rheumatol 27 (2013) 391–404. [9] V. Oke, M. Wahren-Herlenius, Cutaneous lupus erythematosus: clinical aspects and molecular pathogenesis, J. Intern. Med. 273 (2013) 544–554. [10] M.A. Vera-Recabarren, M. García-Carrasco, M. Ramos-Casals, C. Herrero, Comparative analysis of subacute cutaneous lupus erythematosus and chronic cutaneous lupus erythematosus: clinical and immunological study of 270 patients, Br. J. Dermatol 162 (2010) 91–101. [11] A. Kuhn, D. Bein, G. Bonsmann, The 100th anniversary of lupus erythematosus tumidus, Autoimmun. Rev. 8 (2009) 441–448. [12] T.T. Provost, Lupus band test, Int. J. Dermatol. 20 (1981) 475–481. [13] M.V. Dahl, Usefulness of direct immunofluorescence in patients with lupus erythematosus, Arch. Dermatol. 119 (1983) 1010–1017.
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