Soluble granzyme B and cytotoxic T lymphocyte activity in the pathogenesis of systemic lupus erythematosus

doi:10.1016/j.cellimm.2011.03.004

Cellular Immunology

Volume 269, Issue 1, 2011, Pages 16-21

https://doi.org/10.1016/j.cellimm.2011.03.004 Get rights and content

Abstract

Activated cytotoxic T lymphocyte (CTL) mediated target cell death has been implicated in the development of systemic autoimmune disease like SLE. However, the role of soluble granzyme B and its relationship with CTL activity and disease activity is still unknown. In this study, we evaluated role of soluble granzyme B and cytotoxic T lymphocyte activity in SLE patients. The soluble granzyme B was measured in the serum by an enzyme-linked immunosorbent assay while cytotoxic T lymphocyte activity was measured by flow cytometry. The disease activity was determined by using SLE Disease Activity Index (SLEDAI) score. Cytotoxic T lymphocyte activity was increased and strongly associated with disease activity. The soluble granzyme B levels were higher in SLE patients and associated with various clinical features like reduced complement components; C3 & C4 and skin lesion. The soluble granzyme B levels were also sturdily related with severity of the disease. The findings of this study suggest that excessive secretion of soluble granzyme B and enhanced activity of cytotoxic T lymphocyte may play a vital role in the pathogenesis of SLE and organ damage. Also, evaluation of soluble granzyme B may be helpful in monitoring the clinical features associated with activated CTL in SLE.

Highlights

► Increased levels of soluble granzyme B were associated with various clinical parameters like decreased level of complement proteins; C3 & C4. ► Increased levels of soluble granzyme B were associated with skin lesion in SLE patients. ► Activated cytotoxic T lymphocytes were associated with severity of disease in SLE patients. ► Excessive secretion of soluble granzyme B and enhanced activity of CTL may play a vital role in the pathogenesis of SLE and organ damage. ► Also, evaluation of soluble granzyme B may be helpful in monitoring the clinical features associated with activated CTL in SLE.

Introduction

Systemic lupus erythematosus is a multi-system autoimmune disease characterized by the presence of autoantibodies, especially against nuclear components. A key issue in the pathogenesis of lupus is how intracellular antigens become exposed and targeted by the immune system. The heterogeneous nature of disease suggests that numerous factors may be involved in generating the auto-antigens that are associated with SLE. Lymphocyte apoptosis has been invoked in this regard. This is supported by the observations that SLE patients show increased apoptosis of PBMCs, especially T lymphocytes [1], [2], and decreased clearance of activated T cells [3]. A defect in control of apoptosis and delayed clearance of apoptotic cells may provide sustained interaction between reactive oxygen species and apoptotic cell macromolecules generating neoepitopes resulting in autoantibody formation in autoimmune diseases [4].

There is growing evidence that structural changes of auto-antigens during cytotoxic lymphocyte granule-mediated cell death by perforin and granzyme B is a dominant feature of SLE [5]. Granzyme B is a serine protease found in the cytoplasmic granules of CTLs and natural killer (NK) cells that plays an important role in inducing apoptotic changes in target cells during granule exocytosis-induced cytotoxicity. When granzyme B is secreted into the cytoplasm of target cell through the pore formed by perforin, it triggerscytotoxic induced cell death [6], [7], [8].

Recent study shows that granzyme B cleaves auto-antigens that are targeted across the spectrum of systemic autoimmune disease, producing unique auto-antigen fragments that are not seen during caspase-mediated or other forms of cell death. These reports highlight the role of granzyme B as a major contributor to the generation of novel, immunogenic epitopes in autoimmune disease [9], [10]. The role of CD8⁺ cytotoxic T lymphocyte as a generation of auto-antigen in SLE patients is well documented in the previous studies. The histological studies on discoid lupus erythematosus reveal that skin lesions is predominantly composed of T lymphocytes, with a slight predominance of CD4⁺ over CD8⁺ T cells [11], [12].

Until recently, Granzyme B was widely studied at intracellular level, especially in context with apoptosis in SLE [13]. However, the granzymes were originally identified as both intracellular and extracellular proteases, and over the past few years, vast research aims on extracellular Granzyme B activity in autoimmune diseases [14], [15]. The extracellular granzyme B has been reported to associate with the pathogenesis of rheumatoid disease [16], however its association in the pathogenesis of SLE is still to unclear. The aim of this study was to explore the association of an extracellular granzyme B in serum and an intracellular granzyme B expression in CD8⁺ T cells with respect to various clinical features and with disease severity of SLE, which may have further implication in better understanding of the pathogenesis of SLE and for its diagnosis.

Section snippets

Patients and control

Patients for the study were selected from individuals attending out-patient Department of Internal Medicine at Postgraduate Institute of Medical Education and Research, Chandigarh, India. The study included 25 patients with SLE (21 females, 4 males) with mean age of 28.5 ± 7.50 years and the control group consisted of 25 healthy volunteers (21 females, 4 males) with mean age of 26.90 ± 8.0 years. The disease was diagnosed using the American College of Rheumatology (ACR) 1997 revised criteria [17].

Results

The demographic and clinical characteristics of SLE patients and healthy controls are summarized in the Table 1, Table 2. Disease activity was determined by using SLE Disease Activity Index (SLEDAI) score (maximum score of 105): Mild score < 10; Moderate score 10–20; Severe score > 20. Six patients had moderate SLEDAI score while the rest 19 had severe SLEDAI score in the present study. All the patients included in the present study were non-smokers and non-alcoholics, not associated with any other

Discussion

Systemic lupus erythematosus is a multifactorial autoimmune disease characterized by the presence of autoantibodies, especially against nuclear components. The assortment of autoantibodies produced is broad and as a consequence the manifestations of the disease are diverse. A key issue in the pathogenesis of lupus is how intracellular antigens become exposed and targeted by an immune system. Recently, CD8⁺ cytotoxic T lymphocyte has been pleaded as a potent mediator for the generation of

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the grant received from Council of Scientific and Industrial Research, New Delhi, India. We acknowledge Dr. Aman Sharma for helping us to analyse the clinical details of the patients, Dr. Narendra Kumar for the statistical analysis of the data, Mrs. Bhupinder and Mrs. Sandhya for flow cytometry analysis of the samples.

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IL-1β and IL-17 in cutaneous lupus erythematous skin biopsies: could immunohistochemicals indicate a tendency towards systemic involvement?
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Only a fraction of patients with cutaneous lupus erythematosus (CLE) will eventually progress toward systemic disease (SLE).
To find inflammatory biomarkers which could predict the progression of cutaneous lupus erythematosus (CLE) into systemic lupus erythematosus (SLE) using immunohistochemical (IHC) assays.
Immunohistochemical markers for cytotoxic, inflammatory, and anti-inflammatory responses and morphometric methods were applied to routine paraffin sections of skin biopsies, taken from lesions of 59 patients with discoid lupus, subacute lupus, and lupus tumidus. For the diagnosis of SLE, patients were classified by both the American College of Rheumatology (ACR-82) and the Systemic Lupus International Collaborating Clinics (SLICC-12) systems.
Skin samples from CLE/SLE + patients presented higher expression of IL-1β (ARC-82: p = 0.024; SLICC-12: p = 0.0143) and a significantly higher number of cells marked with granzyme B and perforin (ARC: p = 0.0097; SLICC-12: p = 0.0148). Biopsies from CLE/SLE- individuals had higher expression of IL-17 (ARC-82: p = 0.0003; SLICC-12: p = 0.0351) and presented a positive correlation between the density of granzyme A + and FoxP3+ cells (ARC-82: p = 0.0257; SLICC-12: p = 0.0285) and CD8+ cells (ARC-82: p = 0.0075; SLICC-12: p = 0.0102), as well as between granulysin-positive and CD8+ cells (ARC-82: p = 0.0024; SLICC-12: p = 0.0116).
Patients were evaluated at a specific point in their evolution and according to the presence or not of systemic disease. The authors cannot predict how many more, from each group, would have evolved towards SLE in the following years.
In this cohort, immunohistochemical findings suggested that patients with a tendency to systemic disease will show strong reactivity for IL-1β, while those with purely cutaneous involvement will tend to express IL-17 more intensely.
Beyond target cell death – Granzyme serine proteases in health and disease
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Citation Excerpt :
Studies showing the up-regulation of granzymes in human autoimmune diseases have mostly been correlative, and a causal role in immunopathology has not been demonstrated. For example, soluble GzmB, but not K and M, is elevated in SLE patients and correlates with disease severity (Kok et al., 2017; Shah et al., 2011). It was previously shown that cleavage of autoantigens by GzmB is involved in the generation of SLE-specific autoantigens (Graham and Utz, 2005).
Granzymes are a family of small (∼32 kDa) serine proteases with a range of substrate specificities that are stored in, and released from, the cytoplasmic secretory vesicles (‘granules’) of cytotoxic T lymphocytes and natural killer cells. Granzymes are not digestive proteases but finely tuned processing enzymes that target their substrates in specific ways to activate various signalling pathways, or to inactivate viral proteins and other targets. Great emphasis has been placed on studying the pro-apoptotic functions of granzymes, which largely depend on their synergy with the pore-forming protein perforin, on which they rely for penetration into the target cell cytosol to access their substrates. While a critical role for granzyme B in target cell apoptosis is undisputed, both it and the remaining granzymes also influence a variety of other biological processes (including important immunoregulatory functions), which are discussed in this review. This includes the targeting of many extracellular as well as intracellular substrates, and can also lead to deleterious outcomes for the host if granzyme expression or function are dysregulated or abrogated. A final important consideration is that granzyme repertoire, biochemistry and function vary considerably across species, probably resulting from the pressures applied by viruses and other pathogens across evolutionary time. This has implications for the interpretation of granzyme function in preclinical models of disease.
B cell depletion in murine lupus using cytotoxic T lymphocytes in vivo: Feasibility and benefit
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Additionally, low dose rIL-2 therapy in conjunction with ag-specific CD8 CTL promotion could overcome IL-2 defects intrinsic to SLE CD4 Th cells. Although CD8 CTL can play a role in the elimination of autoreactive B cells, enhanced CTL function may contribute to lupus pathology, particularly cutaneous manifestations such as discoid lupus [31]. Thus, specific enhancement of only CTL targeting autoreactive B cells e.g., CAR-T cells is preferable to the use of non-specific CTL activation.
Given the promising results in human lupus with B cell depletion, we tested whether in vivo cytotoxic T lymphocyte (CTL) could eliminate autoreactive B cells in the setting of murine lupus. Using the parent-into-F1 (P → F1) model to generate CTL that eliminate B cells, we found that transfer of NZB parental splenocytes into lupus-prone female NZB/W F1 mice resulted in profound B cell reduction whereas NZW → F1 mice exhibited defective B cell elimination. Using pre-disease or early disease B/W mice as hosts, NZB → F1 mice exhibited B cell depletion and improved proteinuria but no improvement in survival whereas NZW → F1 mice had significantly reduced proteinuria and prolonged survival. Thus, despite the defective IL-2 environment in B/W F1 mice, generation of CTL and B cell depletion is feasible in NZB → F1 mice. The surprising increase in survival for NZW → F1 mice despite defective B cell elimination suggests that NZW splenocytes may contain a beneficial down regulatory cell.
IL-10 producing regulatory and helper T-cells in systemic lupus erythematosus
2019, Seminars in Immunology
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Indeed, the rather surprising finding that anti-IL-10 antibody treatment in SLE rapidly reduced cutaneous manifestations in patients that maintained high levels of autoantibodies [41], suggests that IL-10 might play a pro-inflammatory role in tissues independently of its effects on B-cells. Notably, also CD8+ T-cells can be stimulated by IL-10 [46–48](see review in this special issue by M. Oft), and they might play a pathogenic role in SLE as well [49–51]. Conversely, IL-10 has potent inhibitory effects on monocytes and dendritic cells (DC) [52], but this inhibitory function seems to be intact in SLE [53,54].
Systemic lupus erythematosus (SLE) is a highly heterogeneous autoimmune disease characterised by the production of pathogenic autoantibodies against nuclear self-antigens. The anti-inflammatory and tolerogenic cytokine Interleukin-10 appears to play a paradoxical pathogenic role in SLE and is therefore currently therapeutically targeted in clinical trials. It is generally assumed that the pathogenic effect of IL-10 in SLE is due to its growth and differentiation factor activity on autoreactive B-cells, but effects on other cells might also play a role. To date, a unique cellular source of pathogenic IL-10 in SLE has not been identified. In this review, we focus on the contribution of different CD4⁺T-cell subsets to IL-10 and autoantibody production in SLE. In particular, we discuss that IL-10 produced by different subsets of adaptive regulatory T-cells, follicular helper T-cells and extra-follicular B-helper T-cells is likely to have different effects on autoreactive B-cell responses. A better understanding of the role of IL-10 in B-cell responses and lupus would allow to identify the most promising therapies for individual SLE patients in the future.
Serum levels of soluble Fas ligand, granzyme B and cytochrome c during adjuvant chemotherapy of breast cancer
2015, Clinica Chimica Acta
Citation Excerpt :
Granzyme B is a serine protease normally produced by natural killer cells and cytotoxic T lymphocytes (CTLs) that is released from intracellular granules in response to various stimuli including abnormally proliferating cells [29]. The increased concentrations of circulating granzyme B has been described previously in systemic lupus erythematosus and rheumatoid arthritis [18,19]. In the present study, to the best of our knowledge, we have reported for the first time that significantly increased concentrations of granzyme B in sera of stage II and stage III breast cancer patients before chemotherapy as compared to healthy controls (P < 0.0001).
Anticancer agents used in chemotherapy for tumors induce apoptosis in malignant cells. Soluble Fas ligand, granzyme B and cytochrome c are key elements in the process of apoptosis. The objective of this preliminary study was to evaluate the changes in the serum concentrations of these parameters in breast cancer patients undergoing adjuvant chemotherapy.
Sixty patients with histopathologically proven breast cancer were included in the present study. The blood samples were taken after surgery before chemotherapy and after 3 weeks of administration of the first cycle of chemotherapy. Thirty healthy female controls were selected for comparison. Soluble FasL, granzyme B and cytochrome c were estimated from serum by ELISA.
Significantly increased concentrations of soluble FasL, granzyme B and cytochrome c were found in stage II and stage III of breast cancer patients after chemotherapy compared with concentrations before chemotherapy (P < 0.0001). A significant positive correlation was found between soluble FasL and cytochrome c as well as between granzyme B and cytochrome c in breast cancer patients after chemotherapy.
Serum concentrations of apoptotic markers such as soluble FasL, granzyme B and cytochrome c were increased after administration of the first cycle of chemotherapeutic drugs. The measurement of these circulating apoptotic markers may help clinicians in evaluating treatment efficacy in breast cancer.
Human granzymes: Related but far apart
2014, Medical Hypotheses
Citation Excerpt :
They are part of a family of related serine proteases (EC 3.4.21) where serine serves as the nucleophilic amino acid at the enzyme’s active site [8]. While the majority of GZMs seem to exert their effect locally by crossing the immunological synapse (from the effector cell to the target cell) there are also soluble GZMs found in the extracellular fluid [9–11]. Though the biological effects of these soluble GZMs are not clear, it has been proposed that they may contribute to the degradation of extracellular matrices such as cartilage, as seen in rheumatoid arthritis (RA), and have antibacterial and antiviral activities [12–15].
Granzymes (GZMs) are a class of serine protease, found in cytoplasmic granules of cytotoxic T cells and natural killer cells. The main function of these proteins has been recognized as wiping out viral infections via inducing the apoptosis.
This review will highlight inter and intra species differences of GZMs in terms of their functional and structure. These futures may help to device a strategy for isolation of human specific GZMs, which are needed for understating of their role in immune system and devising an effective immune therapy.

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Soluble granzyme B and cytotoxic T lymphocyte activity in the pathogenesis of systemic lupus erythematosus

Abstract

Highlights

Introduction

Section snippets

Patients and control

Results

Discussion

Conflict of interest

Acknowledgments

Clin. Immunol.

Immunity

Microbes Infect.

Clin. Immunol. Immunopathol.

Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus

J. Immunol.

High levels of circulating early apoptic peripheral blood mononuclear cells in systemic lupus erythematosus

Lupus

Oxygen free radicals and systemic autoimmunity

Clin. Exp. Immunol.

Sources of autoantigens in systemic lupus erythematosus

Curr. Opin. Rheumatol.

Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo

Nat. Rev. Immunol.

Granzyme B: a natural born killer

Immunol. Rev.