Benjamin F. Chong

The Vast Majority of CLA + T Cells Are Resident in Normal Skin

There are T cells within normal, noninflamed skin that most likely conduct immunosurveillance and are implicated in the development of psoriasis. We isolated T cells from normal human skin using both established and novel methods. Skin resident T cells expressed high levels of CLA, CCR4, and CCR6, and a subset expressed CCR8 and CXCR6. Skin T cells had a remarkably diverse TCR repertoire and were mostly Th1 memory effector cells with smaller subsets of central memory, Th2, and functional T regulatory cells. We isolated a surprising number of nonexpanded T cells from normal skin. To validate this finding, we counted T cells in sections of normal skin and determined that there are ∼1 × 106 T cells/cm2 normal skin and an estimated 2 × 1010 T cells in the entire skin surface, nearly twice the number of T cells in the circulation. Moreover, we estimate that 98% of CLA+ effector memory T cells are resident in normal skin under resting conditions. These findings demonstrate that there is a large pool of memory T cells in normal skin that can initiate and perpetuate immune reactions in the absence of T cell recruitment from the blood.

E-Selectin, Thymus- and Activation-Regulated Chemokine/CCL17, and Intercellular Adhesion Molecule-1 Are Constitutively Coexpressed in Dermal Microvessels: A Foundation for a Cutaneous Immunosurveillance System

The success of the cutaneous immune system reflects its ability to rapidly and efficiently recruit leukocytes to areas of trauma and infection. Skin-homing memory T cells expressing cutaneous lymphocyte-associated Ag tether on the walls of postcapillary venules in inflamed skin via interaction with endothelial E-selectin and roll in response to the shear stress imparted by flowing blood. Rolling cells sample the vascular surface for chemoattractant compounds (e.g., thymus- and activation-regulated chemokine/CCL17 interacting with CCR4 on the leukocyte surface) and, if successfully stimulated, progress to firm arrest and transmigration mediated by LFA-1 and vascular ICAM-1. Although it is established that this sequence of events draws T cells into inflamed skin, the mechanisms directing trafficking of T cells to noninflamed skin are less well characterized. We hypothesized that basal expression and colocalization of E-selectin, chemokine (e.g., CCL17), and ICAM-1 in dermal vessels could serve to recruit T cells to noninflamed human skin. Immunohistochemical staining for E-selectin and CD31 demonstrated E-selectin expression in a restricted subset of dermal vessels in noninflamed human skin from three different sites. Confocal multicolor immunofluorescence imaging revealed a nonuniform distribution of E-selectin in dermal vessels as well as colocalization of E-selectin with CCL17 and ICAM-1. Coexpression of these molecules on blood vessels in noninflamed skin provides the basis for a model of cutaneous immunosurveillance system active in the absence of pathologic inflammation.

Risk factors for ANA positivity in healthy persons

IntroductionThe finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.MethodsBiological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles.ResultsOverall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature.ConclusionsRisks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.

Immune function abnormalities in peripheral blood mononuclear cell cytokine expression differentiates stages of cutaneous T-cell lymphoma/mycosis fungoides

Purpose: Mycosis fungoides (MF) is a cutaneous T-cell lymphoma (CTCL) characterized by neoplastic skin-homing T cells. To better understand the immunopathogenesis of MF, we analyzed the functional ability of peripheral blood mononuclear cells (PBMC) from early and late MF/CTCL patients to express cytokine genes. In late stage MF/CTCL, patients were separated into those with blood involvement (+B) and without blood involvement (−B). Experimental Design: We analyzed TH1 (interleukin 2 (IL-2), IFN-γ), TH2 (IL-4, IL-5, IL-10, IL-13), and TH17 (IL-17) cytokine gene expression from activated PBMCs from normal (n = 12), psoriasis (n = 6), early MF/CTCL (n = 11), and late MF/CTCL+B (n = 4) and MF/CTCL−B (n = 3) by quantitative real-time PCR. Results: PBMCs from early MF/CTCL and psoriasis showed higher induction of IL-2, IL-4, and IFN-γ genes than those from normal and late MF/CTCL−B and MF/CTCL+B (P < 0.05) in descending order. PBMCs from late MF/CTCL−B exhibited generally the highest level of IL-5, IL-10, IL-13, and IL-17 expression compared with the other groups. PBMCs from early MF/CTCL and late MF/CTCL−B had similarly elevated IL-13 and IL-17. Of all groups, PBMCs from late MF/CTCL+B had the lowest levels of IL-2 (P < 0.05), IL-4, IFN-γ, IL-13, and IL-17. Conclusions: The different pattern of cytokine gene expression suggests a change in immune function in MF/CTCL from early MF/CTCL to late MF/CTCL−B to late MF/CTCL+B. These stages are consistent with localized disease associated with an anti-tumor immune response and late MF/CTCL associated with a loss of immune function mediated by malignant T cells that share regulatory T cell–like properties.

Dysregulated expression of CXCR4/CXCL12 in subsets of patients with systemic lupus erythematosus.

OBJECTIVE CXCR4 is a chemokine with multiple effects on the immune system. In murine lupus models, we demonstrated that monocytes, neutrophils, and B cells overexpressed CXCR4 and that its ligand, CXCL12, was up-regulated in diseased kidneys. We undertook this study to determine whether CXCR4 expression was increased in peripheral blood leukocytes from patients with systemic lupus erythematosus (SLE) and whether CXCL12 expression was increased in kidneys from patients with SLE. METHODS Peripheral blood leukocytes from 31 SLE patients, 8 normal controls, and 9 patients with rheumatoid arthritis were prospectively analyzed by flow cytometry for CXCR4 expression. Biopsy samples (n = 14) from patients with lupus nephritis (LN) were immunostained with anti-CXCL12 antibody. RESULTS CD19+ B cells and CD4+ T cells from SLE patients displayed a >2-fold increase (P = 0.0001) and >3-fold increase (P < 0.0001), respectively, in median CXCR4 expression compared with that in controls (n = 7-8). Moreover, CXCR4 expression on B cells was 1.61-fold higher in patients with SLE Disease Activity Index (SLEDAI) scores >10 (n = 8) than in patients with SLEDAI scores ≤10 (n = 16) (P = 0.0008), 1.71-fold higher in patients with class IV LN (n = 5) than in patients with other classes of LN (n = 7) (P = 0.02), and 1.40-fold higher in patients with active neuropsychiatric SLE (NPSLE) (n = 6) than in patients with inactive NPSLE (n = 18) (P = 0.01). CXCL12 was significantly up-regulated in the tubules and glomeruli of kidneys in patients with LN (n = 14), with the percentage of positive cells correlating positively with the severity of LN. CONCLUSION CXCR4 appears to be up-regulated in multiple leukocyte subsets in SLE patients. The heightened expression of CXCR4 on B cells in active NPSLE and of CXCL12 in nephritic kidneys suggests that the CXCR4/CXCL12 axis might be a potential therapeutic target for SLE patients with kidney and/or central nervous system involvement.

Targeting the CXCR4/CXCL12 axis in systemic lupus erythematosus

Background: CXCR4 antagonists have garnered much interest as promising treatments for cancer metastases and HIV. Given its ability to attract multiple leukocyte subsets and stimulate B cell production and myelopoeisis, recent attention has been directed to these inhibitors in the treatment of autoimmune diseases, such as systemic lupus erythematosus (SLE). Objective: To assess the potential of CXCR4 antagonists in SLE. Methods: We reviewed literature on the expression of CXCR4 and its ligand CXCL12, and the effects of CXCR4 antagonists in murine and human SLE. Results/conclusions: CXCR4 and CXCL12 have been found at abundant levels in peripheral blood leukocyte subsets as well as immune and non-immune organs in lupus-prone murine models. While SLE patients have displayed upregulated, downregulated, or unchanged levels of CXCR4 in circulating blood lymphocytes, CXCR4 and CXCL12 were found prominently in the skin and kidney, suggesting that the ultimate destinations of CXCR4+ cells include these areas. CXCR4 antagonists have been explored in murine lupus models, in which disease severity and nephritis significantly improved. While clinical trials of CXCR4 antagonists in SLE have yet to be initiated, these inhibitors appear to have the potential to improve disease prognosis in severe lupus patients, particularly those with lupus nephritis.

Determining risk factors for developing systemic lupus erythematosus in patients with discoid lupus erythematosus

Up to 28% of patients with discoid lupus erythematosus (DLE) are susceptible to developing systemic lupus erythematosus (SLE). To better characterize patients with DLE who have a higher potential of developing SLE, we reviewed studies contrasting, firstly, DLE‐only patients (i.e. patients with DLE without SLE) and SLE patients with DLE (i.e. patients who are diagnosed with SLE and DLE simultaneously, and patients with SLE who later develop DLE), and secondly, DLE‐only patients and patients with DLE who progress to SLE. These studies have commonly identified various clinical and laboratory indicators, such as widespread DLE lesions, arthralgias/arthritis, nail changes, anaemia, leucopenia, high erythrocyte sedimentation rates (ESRs) and high titres of antinuclear antibodies (ANAs), which are associated with progression to SLE in patients with DLE, and SLE patients with DLE. Limitations of these studies include inadequate follow‐up time, small numbers of patients with DLE converting to SLE, outdated criteria for SLE diagnosis and retrospective study designs. However, because of the risk of SLE development in patients with DLE, complete skin examinations, joint assessments and laboratory tests including ANA, ESR and full blood counts should be performed regularly for patients with DLE. A prospective study following patients with DLE who do or do not develop SLE is currently underway through the Cutaneous Lupus Registry at the University of Texas Southwestern Medical Center. It will seek to identify clinical features and biomarkers that improve our assessment of risk of systemic spread in these patients.

A multicentre, cross-sectional study on quality of life in patients with cutaneous lupus erythematosus.

Background  A study at the University of Pennsylvania (UPenn) Medical Center demonstrated that quality of life in patients with cutaneous lupus erythematosus (CLE) is negatively impacted. Whether patients with CLE in other geographic locations have similar quality of life is unknown.

Photosensitivity in cutaneous lupus erythematosus

Ultraviolet radiation (UVR) is a well‐known exacerbating factor for cutaneous lupus erythematosus (CLE), with photosensitivity comprising one of the American College of Rheumatology (ACR) diagnostic criteria for systemic lupus erythematosus (SLE). However, discerning true photosensitivity in this population is difficult due to the broad language utilized by the ACR and the delayed‐onset nature of photosensitive lupus lesions.

Differential expression of BAFF and its receptors in discoid lupus erythematosus patients

BACKGROUND B-cell activating factor of the TNF family (BAFF) promotes the maturation and survival of B cells. Because BAFF levels are elevated in systemic lupus erythematosus (SLE) patients, BAFF has been the target of emerging therapies for SLE, such as belimumab. Levels of BAFF and its receptors in discoid lupus erythematosus (DLE) patients are unknown. OBJECTIVE To compare skin and blood mRNA and protein levels of BAFF and its receptors BAFF-R, TACI, and BCMA in DLE subjects with (DLE+/SLE+ (N=28)) and without SLE (DLE+/SLE- (N=35)), psoriasis subjects (N=11), and normal subjects (N=42). METHODS We used quantitative real-time PCR to measure blood and skin BAFF, BAFF-R, TACI, and BCMA mRNA, sandwich ELISAs to measure sera BAFF, and immunohistochemistry to evaluate BAFF and BAFF-R skin protein expression. RESULTS BAFF mRNA and protein levels were highest in DLE+/SLE+blood, followed by DLE+/SLE-, psoriasis, and normal blood. BAFF protein also correlated with anti-nuclear antibodies, and autoantibodies against double-stranded DNA, single-stranded DNA, and ribonucleoprotein, and Systemic Lupus Erythematosus Disease Activity Index scores in DLE patients. While showing no difference between DLE+/SLE+ and DLE+/SLE- skin, BAFF and its receptors mRNA were up-regulated in DLE skin vs. normal and psoriasis skin. DLE skin had higher percentages of BAFF-R⁺ inflammatory cells, likely T cells and macrophages, than psoriasis and normal skin. CONCLUSIONS BAFF may be a serologic marker of systemic disease in DLE patients. BAFF and its receptors are elevated in DLE skin, suggesting that targeted therapies against these proteins could treat refractory DLE patients.