Maria Domenica Guarino

GM-CSF and Pregnancy: Evidence of Significantly Reduced Blood Concentrations in Unexplained Recurrent Abortion Efficiently Reverted by Intravenous Immunoglobulin Treatment

Problem: Certain Th‐2 cytokines and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) are propitious for the success of pregnancy and recurrent spontaneous abortion (RSA) is often characterized by a failure of Th‐2 type responses. These considerations as well as the use of intravenous immunoglobulin (IVIg) in RSA induced us to evaluate the levels of GM‐CSF in normal pregnancies, in pregnant women affected with unexplained RSA and the effects of IVIg treatment.

Anti-thyroid antibodies and antiphospholipid syndrome: evidence of reduced fecundity and of poor pregnancy outcome in recurrent spontaneous aborters.

Problem:  To determine the presence of anti‐thyroid antibodies in patients with primary antiphospholipid syndrome (APS) [antiphospholipid antibodies (aPL) + recurrent spontaneous abortion (RSA)], compare APS alone with APS and thyroid autoimmunity for fecundity and for pregnancy outcome.

Restoration of peripheral blood natural killer and B cell levels in patients affected by rheumatoid and psoriatic arthritis during etanercept treatment.

Etanercept (ETN) is an anti‐tumour necrosis factor (TNF)‐α agent used in rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Few studies focused on the effects of anti‐TNF‐α on peripheral blood cells. We aimed to evaluate peripheral blood cells in RA and PsA patients during ETN treatment and to explore their relationships with disease activity. RA (n = 82) and PsA (n = 32) patients who started ETN were included into the study and evaluated prospectively before the beginning of ETN therapy and after 14, 22, 54 and 102 weeks. Patients were studied in terms of disease activity score on 28 joints (DAS28), clinical response and laboratory findings. Natural killer (NK) cells, B cells and T cells were characterized by immunophenotyping. Both the RA and the PsA patients showed reduced NK and B cell count before ETN treatment compared with controls. A negative correlation was demonstrated between DAS28 and B cell count in RA patients at baseline. Sustained significant increase of NK and B cells up to normal levels was observed in RA and PsA patients along ETN treatment. Increase of NK cell count was associated with a good–moderate clinical response to ETN in both RA and PsA patients. During ETN treatment peripheral blood NK and B cells levels were restored in RA and PsA patients. Correlations between NK and B cells with disease activity were observed, suggesting that those effects could be mediated by ETN treatment.

Auto-reactions, autoimmunity and psoriatic arthritis.

Evidence from the literature suggests that autoimmune processes may drive features of psoriatic arthritis (PsA). Such hypothesis is supported by the evidence that class I major histocompatibility complex (MHC) genes are associated with susceptibility to develop PsA and auto-reactive cells, such as CD8 T cells, T helper (h) 17 and plasma cells, have been demonstrated in PsA. However, no autoantigens have ever been demonstrated in PsA. The presence of a new autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, has been identified in rheumatoid arthritis (RA) patients. These autoantibodies have been associated with a worse disease progression independent of anti-citrulline antibodies (ACPA). In PsA, anti-CarP antibodies have not been evaluated yet. We aimed at analyzing, for the first time, the anti-CarP antibodies in sera of patients with active PsA who were negative for ACPA in order to explore both their presence and their relationship with disease activity. A total of 70 individuals, 30 patients with diagnosis of PsA (according to CASPAR criteria) and 40 healthy controls (HC) were enrolled. We found significantly increased levels of anti-CarP antibodies in PsA patients compared with HC (P<0.0001). Our findings indicate that anti-CarP antibodies are detectable with high specificity and sensibility in PsA patients suggesting an autoimmune background of PsA. Anti-CarP antibodies can be useful in improving the diagnosis of PsA and are correlated with disease activity.

The autoimmune side of hereditary angioedema: Insights on the pathogenesis

Hereditary angioedema (HAE) is an autosomal dominant disease resulting from the deficiency of C1 inhibitor (C1-INH), a glycosylated serine protease inhibitor that plays a regulatory role in the complement system (CS), the contact system and the intrinsic coagulation cascade. HAE disease severity is highly variable and may be influenced by genetic polymorphisms as well as by other factors, such as gender hormone-mediated effects. In HAE, the potential inadequate clearance of immune-complexes (IC) in the presence of reduced levels of CS components and in turn an excess of IC in the tissues results in inflammatory damage and release of autoantigens that may trigger an autoimmune response. Occasional reports link HAE with autoimmune conditions and only few studies have been conducted on large patient populations with controversial results. Although several immunoregulatory disorders have been documented, the prevalence of defined autoimmune diseases in patients with HAE remains debated. The occurrence of autoimmune conditions in HAE patients may worsen the disease severity enhancing the complexity of the comprehensive care.

Downregulation of immunoglobulin-like transcript-4 (ILT4) in patients with psoriatic arthritis.

Objective The immunoglobulin-like transcript-4 (ILT4) is an inhibitory receptor that modulates the activity of innate immune agents. We determined the expression of ILT4 and analysed the relationship with the expression of costimulatory proteins and tumor necrosis factor-α (TNF-α) production in monocytes from patients with psoriatic arthritis (PsA) starting anti-TNF treatment. Methods Peripheral blood monocytes from 15 healthy controls and from 16 patients with PsA were activated in vitro by CD40 ligand (CD40L) and analyzed for ILT4, CD40, CD80 and CD86 expression, and spontaneous lipopolysaccharide (LPS)-induced TNF-α production by flow cytometry, before and after treatment with adalimumab. Results The percentage of ILT4-negative monocytes was greater in PsA patients compared to controls and negatively correlated with DAS44. Normal monocytes treated with sera of PsA patients showed a reduced expression of ILT4 compared with monocytes exposed to sera from controls. CD40, CD80 and CD86 expression was higher in patients compared to controls. Both spontaneous and LPS-induced TNF-α production was restricted to ILT4-negative monocytes and was greater in PsA patients compared to controls. Finally, twelve weeks-treatment with adalimumab resulted in a significant increase of ILT4 expression and a decrease of costimulatory molecules expression in PsA patients, compared to pre-therapy levels. Conclusions These data support the possibility that changes in the immunophenotype of monocytes play a role in the pathogenesis of PSA. Thus, modulation of the expression of ILT4 may represent an enticing new therapeutic target.

Emotional processes and stress in children affected by hereditary angioedema with C1-inhibitor deficiency: a multicenter, prospective study

BackgroundHereditary angioedema with C1-inhibitor deficiency (C1-INH-HAE) is characterized by recurrent edema of unpredictable frequency and severity. Stress, anxiety, and low mood are among the triggering factors most frequently reported. Impaired regulation and processing of emotions, also known as alexithymia, may influence outcomes. The aim of this study was to confirm the presence of alexithymia and stress in children with C1-INH-HAE, to determine whether they are also present in children affected by other chronic diseases, and to investigate their relationship with C1-INH-HAE severity. Data from children with C1-INH-HAE (n = 28) from four reference centers in Italy were compared with data from children with type 1 diabetes (T1D; n = 23) and rheumatoid arthritis (RA; n = 25). Alexithymia was assessed using the Alexithymia Questionnaire for Children scale; perceived stress was assessed using the Coddington Life Event Scale for Children (CLES-C).ResultsMean age (standard deviation [SD]) in the C1-INH-HAE, T1D, and RA groups was 11.8 (3.3), 11.7 (2.9), and 11.1 (2.6) years, respectively. Mean C1-INH-HAE severity score was 5.9 (2.1), indicating moderate disease. Alexithymia scores were similar among disease groups and suggestive of difficulties in identifying and describing emotions; CLES-C scores tended to be worse in C1-INH-HAE children. C1-INH-HAE severity was found to correlate significantly and positively with alexithymia (p = 0.046), but not with perceived stress. Alexithymia correlated positively with perceived stress.ConclusionsAlexithymia is common in children with chronic diseases. In C1-INH-HAE, it may result in increased perceived stress and act as a trigger of edema attacks. Comprehensive management of C1-INH-HAE children should consider psychological factors.

Expression of immunoglobulin-like transcript 4 as an inhibitory receptor in patients with psoriatic arthritis.

Objectives To investigate the presence of immunoglobulin-like transcript (ILT)4 and costimulatory proteins (CD40, CD80 and CD86), as well as tumour necrosis factor (TNF)-α production in antigen-presenting cells (APCs) from patients with psoriatic arthritis, before and after treatment with the antitumour necrosis factor-α therapy, adalimumab. Methods Peripheral blood monocytes from patients with psoriatic arthritis and healthy controls were cultured with CD40 ligand (CD40L) to stimulate differentiation to APCs. Cell-surface phenotype was analysed via fluorescence-activated cell sorting. Results CD40L-stimulation resulted in significantly more ILT4+ monocytes in cultures from control subjects (n = 21) than those from patients (n = 20). ILT4-positivity on CD40L-stimulated monocytes was negatively correlated with disease activity in patients. Adalimumab treatment resulted in significant increases from baseline in ILT4-positivity, and in decreases in CD40, CD80 and CD86-positivity in monocytes from patients. Conclusion The effect of adalimumab on monocyte surface phenotype may be due to modification of the inflammatory milieu associated with therapy-induced reduction of disease activity in psoriatic arthritis.

THU0514 Evidences of A Nontolerogenic-Proinflammatory Surface and Cytokine Phenotype in Monocytes from Patients with Psoriatric Arthritis

Background The immunoglobulin-like transcript-4 (ILT4) is an inhibitory receptor that modulates the activity of innate immune agents. Furthermore, acquisition of an nontolerogenic-proinflammatory phenotype by monocytes may contribute to enhance inflammation and T-cell activation in chronic inflammatory diseases. [1] Objectives to determine the expression of ILT4 and analysed the relationship with the expression of costimulatory proteins as well as tumor necrosis factor-α (TNFα) production in monocytes from patients with psoriatic arthritis (PsA) starting anti-TNF treatment. Methods Peripheral blood monocytes from twenty patients with moderate to severe PsA and 15 healthy control subjects were cultured in vitro in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and analyzed for the surface expression of innate immune receptor immunoglobulin-like transcript 4 (ILT4), CD40, CD80 and CD86, and CD40L-induced and spontaneous lipopolisaccharide (LPS)-induced TNFα production were evaluated by flow cytometry. Results The expression of ILT4 was significantly lower in monocytes from patients with PsA compared with cells from controls (percentage of positive cells: 40.4±14.5 vs. 68.6±18.9, P<0.001). Pearson correlation analysis showed that the expression of ILT4 on monocytes from patients with moderate to severe PsA was indirectly correlated with the severity of PsA, measured by DAS44-ESR score (r=-0.654, confidence interval= -0.868 to -0.234, P=0.006). Greater expression of CD40, CD80 and CD86 and enhanced production of TNF-a in response to CD40L stimulation were detected in ILT4– monocytes from patients with PsA compared with cells from controls. Finally, twelve weeks-treatment with adalimumab resulted in a significant increase of ILT4 expression, a decrease of costimulatory molecules expression and cytokine production in PsA patients, compared to pre-therapy levels. Conclusions Monocytes of patients with moderate to severe PsA acquire a nontolerogenic-proinflammatory phenotype reverted by anti-TNF treatment. Since monocytes migrate from the peripheral blood into inflamed joints, and GM-CSF acts as a proinflammatory cytokine in different forms of inflammatory arthritis, it is possible to speculate that these phenotypic changes may enhance the antigen presenting ability and the proinflammatory activity of monocytes of PsA patients and then contributes to the autoimmune processes in articular inflammation. This knowledge provides new evidences on the immunomodulatory properties of ILT4 supporting the possibility that changes in the immunophenotype of monocytes play a role in the pathogenesis of PSA. Thus, modulation of the expression of ILT4 may represent an enticing new therapeutic target. References Dietrich J, Cella M, Colonna M. Ig-like transcript 2 (ILT2)/leukocyte Ig-like receptor 1 (LIR1) inhibits TCR signaling and actin cytoskeleton reorganization. J. Immunol. 2001;166:2514-2521 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.1993

AB0092 Downregulation of immunoglobulinlike transcript 4 (ilt4) in patients with rheumatoid arthritis

Background Rheumatoid arthritis (RA) is characterized by destruction of juxtaarticular cartilage and bone due to excessive activation of inflammatory cells of which the underlying mechanisms are not fully elucidated. Macrophages may play a major role in the process of inflammation and tissue destruction since they are the predominant source of interleukin (IL)-1β and tumor necrosis factor (TNF)-α that are central to the pathogenesis of RA. Leukocyte immunoglobulin-like receptors (LILRs) are emerging as critical regulators of the threshold and amplitude of leukocyte activation, and perturbed expression of LILRs may contribute to uncontrolled inflammation (1). In particular, the inhibitory LILR-2, also called the immunoglobulinlike transcript 4 (ILT4), which is predominantly expressed on macrophages, provides inhibitory signals (2). Thus, it is tempting to speculate that decreased ILT4 expression on macrophages in RA may lead to a chronic activation of these cells and thereby result in exaggerated inflammation. Objectives To investigate the expression of ILT4 and the relationship between ILT4 and pro-inflammatory cytokine production in monocytes from RA patients before and after anti-TNF-a therapy. Methods Surface expression of ILT4 and lipopolysaccharide (LPS)-induced TNF-a and IL-1b production were analysed by FACS on circulating monocytes from 13 patients with RA and 10 age and sex matched healthy controls, before and after 12-weeks therapy with anti-TNF-a mAb. Results ILT4 expression was significantly decreased on both CD14+/CD16- and CD14-/CD16+ monocytes from RA patients as compared to controls. In addition, a significant reduction in the ability to upregulate ILT4 surface expression under condition of activation by granulocyte-macrophage colony stimulating factor (GM-CSF) was documented in moncytes from RA patients with respect to cells from controls. Monocytes from both, patients and controls, did not spontaneously produce proinflammatory cytokines. However, after LPS stimulation TNF-a and IL-1b production was significantly increased in patients compared to controls. This increased cytokine response was not due to a enhanced susceptibility of monocytes from RA patients to LPS but rather to an expansion of the ILT4- monocyte subset, since TNF-a and IL-1b expression was almost exclusively observed in this cell subset. Twelve-weeks treatment with anti-TNF-a mAb resulted in a significant increase of monocyte ILT4 expression, ILT4 response to GM-CSF activation and in a significantly reduced proinflammatory cytokine expression, compared with pre-therapy levels. Conclusions The finding that RA patients display a low expression of monocytic ILT4, which may lead to more severe tissue damage and impairment of the control of autoreactive cells by upregulating the proinflammatory and antigen-presenting functions of monocytes, suggests that drugs targeting this molecule may have significant therapeutic value for the management of patients. References Colonna M, et al. J Exp Med. 1997;186:1809-18 Chang CC, et al. Nat Immunol 2002;3:237-43 Disclosure of Interest None Declared