Jonathan D. Jones

Rituximab mediates loss of CD19 on B cells in the absence of cell death

OBJECTIVE To evaluate loss of the B cell-specific marker CD19 after the addition of rituximab (RTX) to healthy donor blood and to determine the role of complement-mediated cytotoxicity in these cells. METHODS Whole blood and peripheral blood mononuclear cells (PBMCs) from healthy donors were evaluated for the loss of CD19 in the presence of RTX using flow cytometry. The effect of complement on CD19 loss was examined using serum-free media, C3- and C5-deficient sera, and a C5-blocking antibody. Evidence of B cell death was evaluated by measuring messenger RNA (mRNA) levels as well as by flow cytometry. Transfer of CD19 antigen to monocytes and neutrophils was evaluated by flow cytometry and confocal microscopy. RESULTS RTX induced a rapid decrease in CD19 count (mean 51%; n = 37) in PBMCs. This reduction occurred in the absence of complement. Despite the decrease in CD19 expression, B cell death did not occur, as evidenced by a lack of change in CD19 or CD20 mRNA levels and a lack of change in CD19 levels determined by intracellular staining and through the use of viability dyes. The CD19 antigen was shown to be transferred to monocytes and neutrophils in an Fc-dependent manner. CONCLUSION Our findings indicate that the addition of RTX to healthy donor PBMCs in vitro results in complement-independent loss of CD19 without causing B cell death. CD19 is transferred from B cells to monocytes and neutrophils during shaving of the RTX-CD20 complex in an Fc-dependent manner. These data suggest that monitoring the effect of RTX by measuring the CD19+ cell count may be compromised by this activity.

The role for neutrophil extracellular traps in cystic fibrosis autoimmunity

While respiratory failure in cystic fibrosis (CF) frequently associates with chronic infection by Pseudomonas aeruginosa, no single factor predicts the extent of lung damage in CF. To elucidate other causes, we studied the autoantibody profile in CF and rheumatoid arthritis (RA) patients, given the similar association of airway inflammation and autoimmunity in RA. Even though we observed that bactericidal permeability-increasing protein (BPI), carbamylated proteins, and citrullinated proteins all localized to the neutrophil extracellular traps (NETs), which are implicated in the development of autoimmunity, our study demonstrates striking autoantibody specificity in CF. Particularly, CF patients developed anti-BPI autoantibodies but hardly any anti-citrullinated protein autoantibodies (ACPA). In contrast, ACPA-positive RA patients exhibited no reactivity with BPI. Interestingly, anti-carbamylated protein autoantibodies (ACarPA) were found in both cohorts but did not cross-react with BPI. Contrary to ACPA and ACarPA, anti-BPI autoantibodies recognized the BPI C-terminus in the absence of posttranslational modifications. In fact, we discovered that P. aeruginosa-mediated NET formation results in BPI cleavage by P. aeruginosa elastase, which suggests a novel mechanism in the development of autoimmunity to BPI. In accordance with this model, autoantibodies associated with presence of P. aeruginosa on sputum culture. Finally, our results provide a role for autoimmunity in CF disease severity, as autoantibody levels associate with diminished lung function.

The causes of drug-induced muscle toxicity.

Purpose of reviewClinically identified myopathies are frequently a consequence of medication toxicities. However, recognizing drug-induced myopathies is sometimes difficult. Developing a greater understanding of the underlying mechanisms of drug-induced muscle toxicity will promote enhanced awareness and recognition, and improved management of these syndromes. Recent findingsThe adverse impact of certain drugs on muscle metabolism, muscle cell atrophy, and myocyte apoptosis is increasingly clear. Glucocorticoids impair glucose handling and directly promote protein catabolism. Statins impair mitochondrial function and alter intracellular signaling proteins, which can lead to myocyte apoptosis. Alternatively, statins can induce an autoimmune necrotizing myositis. Several medications impair autophagy, thus limiting access to the needed glycogen stores. SummaryThis review provides an overview of the main underlying mechanisms of drug-induced myopathies. These myopathies will most often be related to a drugs ability to alter metabolism and protein balance, induce necrosis, or impair autophagy.

Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels

IntroductionWe hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity.MethodsSerum from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, rheumatoid factor (RF) levels, anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); other parameters were obtained by chart review. A confirmatory analysis was performed using samples from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort. The Wilcoxon rank-sum test, a t-test and Spearman’s correlation analysis were utilized to determine relationships between variables.ResultsIn both the Dartmouth and Sherbrooke cohorts, CXCL13 levels were selectively increased in seropositive relative to seronegative RA patients (P = 0.0002 and P < 0.0001 for the respective cohorts), with a strong correlation to both immunoglobulin M (IgM) and IgA RF levels (P < 0.0001). There was a weaker relationship to ACPA titers (P = 0.03 and P = 0.006, respectively) and total IgG (P = 0.02 and P = 0.14, respectively). No relationship was seen with regard to age, sex, shared epitope status or inclusion high-sensitivity C-reactive protein (hsCRP) in either cohort or regarding the presence of baseline erosions in the Sherbrooke Cohort, whereas a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) was seen in the Dartmouth cohort but not the Sherbrooke cohort.ConclusionUsing both established and early RA cohorts, marked elevations of serum CXCL13 levels resided nearly completely within the seropositive population. CXCL13 levels exhibited a strong relationship with RF, whereas the association with clinical parameters (age, sex, DAS28-CRP and erosions) or other serologic markers (ACPA and IgG) was either much weaker or absent. Elevated serum CXCL13 levels may identify a subset of seropositive RA patients whose disease is shaped by or responsive to RF production.

Idiopathic inflammatory myopathies—a review

The idiopathic inflammatory myopathies (IIM) constitute a subset of autoimmune conditions primarily affecting muscle, along with many extra-muscular manifestations. Proximal muscle weakness is the dominant symptom in the IIM. In addition, many patients will have cutaneous manifestations, pulmonary manifestations, and arthritis. There is an increased incidence of malignancy with the IIM. Diagnostic tools beyond history and physical exam include serum muscle enzyme and autoantibody testing, magnetic resonance imaging (MRI) of the extremities, ultrasound, neurologic testing, and muscle biopsy. Several options for treatment are reviewed.

Immunoglobulin G4-related disease in the urinary bladder

Immunoglobulin G4‐related disease is a fibroinflammatory condition of unclear etiology that can present with inflammatory changes and enlargement of a wide variety of organs, most commonly in the gastrointestinal tract. A diagnosis requires an elevated serum immunoglobulin G4 concentration and a tissue biopsy showing a dense plasma cell infiltrate with an increased percentage of immunoglobulin G4+ plasma cells. This disease infrequently presents in the genitourinary tract, and as such might be unfamiliar to and potentially overlooked by urologists. Here we present the third reported case of immunoglobulin G4‐related disease manifesting as a mass in the urinary bladder.

Induction of Interleukin‐6 Production by Rituximab in Human B Cells

Rituximab (RTX) is a chimeric monoclonal antibody specific for human CD20. The utility of CD20 targeting by RTX has revolutionized the treatment of B cell malignancies (1-3). In addition, RTX has found wide application in many autoimmune disorders, such as anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, autoimmune bullous diseases, and rheumatoid arthritis (RA) (4). RTX results in B cell depletion through several effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and induction of apoptosis (5). Interestingly, none of these mechanisms has been able to account for the incomplete relationship between B cell depletion and clinical response in RA (6-8). Thus, it seems reasonable to consider alternative effector functions of RTX that are not associated with B cell depletion (9-12). In this regard, there is increasing appreciation of an alternative effector function of RTX, that of trogocytosis (13, 14). Trogocytosis refers to the process when one cell ‘gnaws’ or ‘shaves’ a portion of another cells membrane following the creation of an immunologic synapse. This phenomenon has been described to occur in many cellular interactions, including antibody/FcγR engagement, T cell receptor/antigen presenting cell engagement, and B cell receptor/cell surface peptide engagement (13, 15-17). RTX mediated trogocytosis occurs when an FcγR bearing cell such as a monocyte engages the Fc portion of B cell-bound RTX and removes the RTX-CD20 complex, as well as a portion of the B cell membrane, without altering B cell viability (13, 14, 18, 19). The clinical significance of RTX mediated trogocytosis still is not fully understood, but holds considerable relevance as there is increased recognition of trogocytosis by several other antibody-based therapeutics, such as epratuzumab, daclizumab, trastuzumab, and cetuximab (20-22). We previously identified that RTX exhibits little or no complement dependent cytotoxicity on normal human B cells (14). Instead, in the presence of RTX, phagocytes mediate rapid trogocytosis of CD19 and CD20 in the absence of cell death. Since the absence of CD19 and CD20 from B cells is associated with immunodeficiency (23, 24), this raised the possibility that RTX-dependent trogocytosis might alter the phenotype and function of B cells. In this model, following trogocytosis, functionally impaired B cells might result from their CD19- or CD20-deficiency states. We tested this hypothesis by characterizing phenotypic and functional responses of B cells following RTX-dependent trogocytosis. Instead of seeing functional impairment of B cells, RTX mediated trogocytosis of human B cells results in a selective and marked increase in production and release of IL-6. As we describe, the effect of RTX on IL-6 production was surprisingly specific both in terms of the cytokines being affected but also in its requirement for trogocytosis. In addition to identifying a novel pathway that triggers IL-6 production by B cells, this observation may be relevant to acute and chronic effects of RTX in shaping the immune response including infusion reactions (6, 7).Rituximab (RTX), an anti‐CD20 monoclonal antibody, is highly effective in the treatment of several autoimmune diseases. The mechanism by which RTX treatment improves rheumatoid arthritis and antineutrophil cytoplasmic antibody–associated vasculitis is not easily related to B cell depletion alone. Prior studies have shown that RTX mediates a rapid stripping of CD20 and CD19 from the human B cell through a process known as trogocytosis. The aim of the present study was to investigate whether changes in B cell phenotype resulting from trogocytosis would diminish the ability of B cells to promote autoimmune disease.

Role for ZAP-70 Signaling in the Differential Effector Functions of Rituximab and Obinutuzumab (GA101) in Chronic Lymphocytic Leukemia B Cells

Rituximab (RTX) has been the hallmark anti-CD20 mAb for the treatment of B cell neoplasms, including B cell chronic lymphocytic leukemia (B-CLL). Recently, a novel humanized anti-CD20 mAb obinutuzumab (GA101) has been implemented as first-line CLL therapy. Treatment of CLL patients with RTX is associated with CD20 loss via an FcγR-mediated process, trogocytosis. RTX-induced trogocytosis has been characterized as both the means of resistance to therapy, via loss of cell surface target proteins (antigenic modulation), as well as a process that alters B cell phenotype and function. This study investigates the nature and clinical relevance of GA101-mediated trogocytosis. In this study, we demonstrate that GA101 is a more potent mediator of trogocytosis than RTX in vitro in both normal B cells and B-CLL cells. Qualitative differences in the effector function of these anti-CD20 Abs appear specific to B-CLL cells. GA101-mediated CD19 and CD20 trogocytosis from B-CLL cells is associated with its ability to induce homotypic adhesion (HA). The degree of HA varies between CLL patients and positively correlates with the expression of ZAP-70, a BCR-associated kinase. Deregulation of ZAP-70 using tyrosine kinase inhibitors, gefitinib or ibrutinib, diminishes HA formation and trogocytosis by GA101. Taken together, these findings elucidate the differences in trogocytosis and HA formation mediated by anti-CD20 mAbs RTX and GA101, as well as provide a novel link between ZAP-70 expression and these effector functions.

NETtling the host: Breaking of tolerance in chronic inflammation and chronic infection.

How and why we break tolerance to self-proteins still remains a largely unanswered question. Neutrophils have been identified as a rich source of autoantigens in a wide array of autoimmune diseases that arise as a consequence of different environmental and genetic factors, e.g. rheumatoid arthritis (RA), lupus, vasculitis, cystic fibrosis (CF) etc. Specifically, neutrophil extracellular trap (NET) formation has been identified as a link between innate and adaptive immune responses in autoimmunity. Autoantigens including neutrophil granular proteins (targeted by anti-neutrophil cytoplasmic antibodies, ANCA) as well as post-translationally modified proteins, i.e. citrullinated and carbamylated proteins targeted by anti-citrullinated protein antibodies (ACPA) and anti-carbamylated protein antibodies (ACarPA), respectively, localize to the NETs. Moreover, NETs provide stimuli to dendritic cells that potentiate adaptive autoimmune responses. However, while NETs promote inflammation and appear to induce humoral autoreactivity across autoimmune diseases, the antigen specificity of autoantibodies found in these disorders is striking. These unique autoantigen signatures suggest that not all NETs are created equal and that the environment in which NETs arise shapes their disease-specific character. In this review article, we discuss the effects of different stimuli on the mechanism of NET formation as well as how they contribute to antigen specificity in the breaking of immune tolerance. Specifically, we compare and contrast the autoreactive nature of NETs in two settings of chronic airway inflammation: one triggered by smoking, a recognized environmental NET stimulus in RA patients, and one mediated by Pseudomonas aeruginosa, the most prevalent lung pathogen in CF patients. Finally, we draw attention to novel findings that, together with the specific environmental/chemical stimuli, should be taken into account when investigating how and why antigen specificity arises in the context of NET formation.

ACPA, ACarPA and Rheumatoid Arthritis: Azurophilic Granules Sing the Blues

Arginine and lysine are distinguished by their identity as the most cationic (basic) amino acids. They are also the target of many post-translational modifications, e.g. acetylation, citrullination, hydroxylation, methylation, ubiquitination. These modifications are catalyzed by a series of enzymes, presumably to enable a tapestry of functionality by a single protein, including regulation of DNA transcription, repair and replication. Besides their functional roles in normal physiological conditions, some of these protein modifications have been recognized as inflammatory triggers and targets of autoimmune reactions. This article is protected by copyright. All rights reserved.