Isolated anti-Ro52 identifies a severe subset of Sjögren's syndrome patients.

Introduction: Serum autoantibodies targeting the SSA/Ro proteins are a key component of the classification criteria for the diagnosis of Sjögren's syndrome (SS). Most patients' serum reacts with both Ro60 and Ro52 proteins. Here we compare the molecular and clinical characteristics of patients diagnosed with SS with anti-Ro52 in the presence or absence of anti-Ro60/La autoantibodies. Methods: A cross-sectional study was performed. Patients in the SS biobank at Westmead Hospital (Sydney, Australia) that were positive for anti-Ro52 were included and stratified based on the absence (isolated) or presence (combined) of anti-Ro60/La, measured by line immunoassay. We examined clinical associations and the serological and molecular characteristics of anti-Ro52 using ELISA and mass spectrometry in serological groups. Results: A total of 123 SS patients were included for study. SS patients with isolated anti-Ro52 (12%) identified a severe serological subset characterised by higher disease activity, vasculitis, pulmonary involvement, rheumatoid factor (RhF) and cryoglobulinaemia. Serum antibodies reacting with Ro52 in the isolated anti-Ro52 subset displayed less isotype switching, less immunoglobulin variable region subfamily usage and a lower degree of somatic hypermutation than the combined anti-Ro52 subset. Conclusions: In our cohort of SS patients, isolated anti-Ro52 represents a severe subset of SS, and is associated with the presence of cryoglobulinaemia. We therefore provide clinical relevance to the stratification of SS patients by their sero-reactivities. It is possible that the autoantibody patterns may be immunological epiphenomena of the underlying disease process, and further work is required to unearth the mechanisms of the differential clinical phenotypes.

Immunoglobulin repertoire restriction characterizes the serological responses of patients with predominantly antibody deficiency.

BACKGROUND: Predominantly antibody deficiency (PAD) is the most common category of inborn errors of immunity and is underpinned by impaired generation of appropriate antibody diversity and quantity. In the clinic, responses are interrogated by assessment of vaccination responses, which is central to many PAD diagnoses. However, the composition of the generated antibody repertoire is concealed from traditional quantitative measures of serological responses. Leveraging modern mass spectrometry-based proteomics (MS-proteomics), it is possible to elaborate the molecular features of specific antibody repertoires, which may address current limitations of diagnostic vaccinology. OBJECTIVES: We sought to evaluate serum antibody responses in patients with PAD following vaccination with a neo-antigen (severe acute respiratory syndrome coronavirus-2 vaccination) using MS-proteomics. METHODS: Following severe acute respiratory syndrome coronavirus-2 vaccination, serological responses in individuals with PAD and healthy controls (HCs) were assessed by anti-S1 subunit ELISA and neutralization assays. Purified anti-S1 subunit IgG and IgM was profiled by MS-proteomics for IGHV subfamily usage and somatic hypermutation analysis. RESULTS: Twelve patients with PAD who were vaccine-responsive were recruited with 11 matched vaccinated HCs. Neutralization and end point anti-S1 titers were lower in PAD. All subjects with PAD demonstrated restricted anti-S1 IgG antibody repertoires, with usage of <5 IGHV subfamilies (median: 3; range 2-4), compared to ≥5 for the 11 HC subjects (P < .001). IGHV3-7 utilization was far less common in patients with PAD than in HCs (2 of 12 vs 10 of 11; P = .001). Amino acid substitutions due to somatic hypermutation per subfamily did not differ between groups. Anti-S1 IgM was present in 64% and 50% of HC and PAD cohorts, respectively, and did not differ significantly between HCs and patients with PAD. CONCLUSIONS: This study demonstrates the breadth of anti-S1 antibodies elicited by vaccination at the proteome level and identifies stereotypical restriction of IGHV utilization in the IgG repertoire in patients with PAD compared with HC subjects. Despite uniformly pauci-clonal antibody repertoires some patients with PAD generated potent serological responses, highlighting a possible limitation of traditional serological techniques. These findings suggest that IgG repertoire restriction is a key feature of antibody repertoires in PAD.

Can C-reactive protein be used as a surrogate marker of IL-6 in a broad array of clinical entities?

Background: C-reactive protein (CRP) is commonly performed, whereas cytokine testing is limited to research. Aims: To determine CRP correlation to cytokines IL-6, IL-1ß and TNF-α. Results: Consecutive samples (n = 307) were collected over 24 h. Ninety-six patients (31%) had acute infections, and 23 patients (7.5%) had autoimmune or inflammatory disease presentations. A strong correlation between CRP and two IL-6 assays (r = 0.74 and r = 0.71; p < 0.001) was present. CRP did not correlate with IL-1ß and TNF-α across the data set. Bacterial infection had a significantly higher CRP and IL-6 (p < 0.001), while only CRP was elevated in inflammatory and autoimmune diseases (p < 0.001). Discussion: CRP may be used as a surrogate marker of IL-6 levels in the routine diagnostic laboratories.

Neutrophils Require Activation to Express Functional Cell-Surface Complement Receptor Immunoglobulin.

The phagocytosis-promoting complement receptor, Complement Receptor Immunoglobulin (CRIg), is exclusively expressed on macrophages. It has been demonstrated that expression in macrophages could be modulated by inflammatory mediators, including cytokines. This raised the possibility that a major phagocyte, the neutrophil, may also express CRIg following activation with inflammatory mediators. Here we show that resting peripheral blood neutrophil lysates subjected to protein analysis by Western blot revealed a 35 kDa CRIg isoform, consistent with the expression of CRIg mRNA by RT-PCR. By flow cytometry, CRIg was detected intracellularly and in very minor amounts on the cell surface. Interestingly, expression on the cell surface was significantly increased to functional levels after activation with inflammatory mediators/neutrophil activators; N-Formylmethionine-leucyl-phenylalanine, tumor necrosis factor (TNF), Granulocyte-Macrophage Colony stimulating Factor (GM-CSF), bacterial lipopolysaccharide, leukotriene B4 and phorbol myristate acetate. The increase in expression required p38 MAP kinase and protein kinase C activation, as well as intracellular calcium. Neutrophils which were defective in actin microfilament reorganization due to a mutation in ARPC1B or inhibition of its upstream regulator, Rac2 lose their ability to upregulate CRIg expression. Inhibition of another small GTPase, Rab27a, with pharmacological inhibitors prevented the increase in CRIg expression, suggesting a requirement for the actin cytoskeleton and exocytosis. Engagement of CRIg on TNF-primed neutrophils with an anti-CRIg monoclonal antibody increased the release of superoxide and promoted the activation of p38 but not ERK1/ERK2 or JNK MAP kinases. The TNF-induced increase in killing of Staphylococcus aureus was blocked by the anti-CRIg antibody. Adding to the anti-microbial role of CRIg, it was found that GM-CSF priming lead to the release of neutrophil extracellular traps. Interestingly in contrast to the above mediators the anti-inflammatory cytokine IL-10 caused a decrease in basal expression and GM-CSF induced increase in CRIg expression. The data demonstrate that neutrophils also express CRIg which is regulated by inflammatory mediators and cytokines. The findings show that the neutrophil antimicrobial function involving CRIg requires priming as a means of arming the cell strategically with microbial invasion of tissues and the bloodstream.

Vitamin D upregulates the macrophage complement receptor immunoglobulin in innate immunity to microbial pathogens.

Vitamin D deficiency remains a global concern. This 'sunshine' vitamin is converted through a multistep process to active 1,25-dihydroxyvitamin D3 (1,25D), the final step of which can occur in macrophages. Here we demonstrate a role for vitamin D in innate immunity. The expression of the complement receptor immunoglobulin (CRIg), which plays an important role in innate immunity, is upregulated by 1,25D in human macrophages. Monocytes cultured in 1,25D differentiated into macrophages displaying increased CRIg mRNA, protein and cell surface expression but not in classical complement receptors, CR3 and CR4. This was associated with increases in phagocytosis of complement opsonised Staphylococcus aureus and Candida albicans. Treating macrophages with 1,25D for 24 h also increases CRIg expression. While treating macrophages with 25-hydroxyvitamin D3 does not increase CRIg expression, added together with the toll like receptor 2 agonist, triacylated lipopeptide, Pam3CSK4, which promotes the conversion of 25-hydroxyvitamin D3 to 1,25D, leads to an increase in CRIg expression and increases in CYP27B1 mRNA. These findings suggest that macrophages harbour a vitamin D-primed innate defence mechanism, involving CRIg.

Delayed Blood Processing Leads to Rapid Deterioration in the Measurement of the Neutrophil Respiratory Burst by the Dihydrorhodamine-123 Reduction Assay.

BACKGROUND: Neutrophils ex vivo in whole blood specimens are widely understood to decay rapidly when compared to other leukocytes, requiring assessment of neutrophil activity to be performed shortly after blood collection. There is a disparity in evidence for decay rates in measurements and recommended time-frames for assaying neutrophil parameters in particular assays following blood collection. We, therefore, evaluated the decline in the neutrophil respiratory burst, typically screened for assessing congenital NADPH oxidase defects, over a shorter time-course than previously published experiments. METHODS: The neutrophil respiratory burst was assessed by flow cytometric detection of DHR-123 oxidation to rhodamine-123 (Rho123), following stimulation of neutrophils by phorbol myristate acetate (PMA), in heparinized healthy donor blood specimens immediately following venipuncture, and then at 3 and 5 h later with ambient temperature or refrigerated specimen storage. RESULTS: A consistent time-dependent decline in the Rho123 fluorescence of PMA-stimulated neutrophils was detected in the healthy donor specimens, indicating a decay in respiratory burst activity. Neutrophil oxidative indexes calculated for half of the specimens at 3 and 5 h of age, fell below our normal laboratory lower limit. We also found that Rho123 histograms of PMA-stimulated neutrophils from stored healthy donor specimens have a risk of misinterpretation due to mimicking the appearance of histograms from carriers of CGD and other NADPH oxidase defects. Refrigeration of specimens did not significantly minimize decay. CONCLUSIONS: DHR assay of the neutrophil respiratory burst from blood specimens at 3 h post-venipuncture and beyond can generate unreliable clinical measurements due to decay. © 2019 International Clinical Cytometry Society.

Human Dendritic Cells Express the Complement Receptor Immunoglobulin Which Regulates T Cell Responses.

The B7 family-related protein V-set and Ig containing 4 (VSIG4), also known as Z39Ig and Complement Immunoglobulin Receptor (CRIg), is the most recent of the complement receptors to be identified, with substantially distinct properties from the classical complement receptors. The receptor displays both phagocytosis-promoting and anti-inflammatory properties. The receptor has been reported to be exclusively expressed in macrophages. We now present evidence, that CRIg is also expressed in human monocyte-derived dendritic cells (MDDC), including on the cell surface, implicating its role in adaptive immunity. Three CRIg transcripts were detected and by Western blotting analysis both the known Long (L) and Short (S) forms were prominent but we also identified another form running between these two. Cytokines regulated the expression of CRIg on dendritic cells, leading to its up- or down regulation. Furthermore, the steroid dexamethasone markedly upregulated CRIg expression, and in co-culture experiments, the dexamethasone conditioned dendritic cells caused significant inhibition of the phytohemagglutinin-induced and alloantigen-induced T cell proliferation responses. In the alloantigen-induced response the production of IFNγ, TNF-α, IL-13, IL-4, and TGF-ß1, were also significantly reduced in cultures with dexamethasone-treated DCs. Under these conditions dexamethasone conditioned DCs did not increase the percentage of regulatory T cells (Treg). Interestingly, this suppression could be overcome by the addition of an anti-CRIg monoclonal antibody to the cultures. Thus, CRIg expression may be a control point in dendritic cell function through which drugs and inflammatory mediators may exert their tolerogenic- or immunogenic-promoting effects on dendritic cells.