Over‐expression of prothymosin‐α antagonizes TGFβ signalling to promote the development of emphysema

Emphysema, a major consequence of chronic obstructive pulmonary disease (COPD), is characterized by the permanent airflow restriction resulting from enlargement of alveolar airspace and loss of lung elasticity. Transforming growth factor‐β (TGFβ) signalling regulates the balance of matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) to control matrix homeostasis. Patients with COPD have dysregulated TGFβ signalling and reduced histone deacetylase (HDAC) activity through epigenetic up‐regulation of histone acetylation in the promoters of pro‐inflammatory genes. However, the potential link between decreased HDAC activity and dysregulated TGFβ signalling in emphysema pathogenesis remains to be determined. Prothymosin α (ProT), a highly conserved acidic nuclear protein, plays a role in the acetylation of histone and non‐histone proteins. The aim of this study was to test the hypothesis that ProT inhibits TGFβ–Smad signalling through Smad7, thereby contributing to emphysema pathogenesis. We show that ProT enhances Smad7 acetylation by decreasing its association with HDAC and thereby down‐regulates TGFβ–Smad signalling. ProT caused an imbalance between MMP and TIMP through acetylated Smad7 in favour of MMP expression. In addition to interfering with R‐Smad activation and targeting receptors for degradation in the cytoplasm, acetylated Smad7 potentiated by ProT competitively antagonized binding of the pSmad2/3–Smad4 complex to the TIMP‐3 promoter, resulting in reduced TIMP‐3 expression. These effects were detected in ProT‐over‐expressing cells, lungs of ProT transgenic mice displaying an emphysema phenotype and in emphysema patients. Importantly, increased Smad7 and reduced TIMP‐3 were found in the lungs of emphysema patients and mice with cigarette smoke extract (CSE)‐induced emphysema. Such effects could be abrogated by silencing endogenous ProT expression. Collectively, our results uncover acetylated Smad7 regulated by ProT as an important determinant in dysregulated TGFβ signalling that contributes to emphysema pathogenesis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Transforming growth factor‐β/Smad ‐ signalling pathway and conjunctival remodelling in vernal keratoconjunctivitis

Background Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characterized by corneal complications and the formation of giant papillae. Sma‐ and Mad‐related proteins (Smad) modulate extracellular matrix gene expression during wound healing, inflammation and tissue remodelling. Objective To investigate the relationship between allergic inflammation and TGF‐β/Smad signalling pathway, expression in VKC patients and in primary cultured conjunctival fibroblasts exposed to mediators found previously over‐expressed in VKC. Methods Smad‐2, ‐3, ‐7, phospho‐(p)Smads, TGF‐β1 and ‐β2 were evaluated in the conjunctiva of normal subjects (CT) and VKC patients by immunohistochemistry. The expression of Smads, pro‐collagen I (PIP), TGF‐β1, ‐β2, mitogen‐activated protein kinase (p38/MAPK), c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK1/2) were also determined in conjunctival fibroblast cultures exposed to histamine, IL‐4, ‐13, TGF‐β1, IFN‐γ and TNF‐α using immunostaining or RT‐PCR. Results Immunostaining for Smad‐2, ‐3, pSmad‐2, ‐3, TGF‐β1, ‐β2 and PIP was significantly increased in VKC stroma compared with CT. In conjunctival fibroblast cultures, Smad‐3 and PIP were stimulated by histamine, IL‐4, ‐13 and TGF‐β1 exposure, while PIP was reduced by IFN‐γ, and TNF‐α mRNA expression of Smad‐3 was increased by histamine, while Smad‐7 was reduced by IL‐4. In addition, histamine, IL‐4 and TNF‐α increased JNK and ERK1/2 expression. Conclusion and Clinical Relevance The TGF‐β/Smad signalling pathway is over‐expressed in VKC tissues and modulated in conjunctival fibroblasts by histamine, IL‐4, TGF‐β1 and TNF‐α. These mechanisms may be involved in fibrillar collagen production, giant papillae formation and tissue remodelling typical of VKC and might provide new therapeutic targets for its treatment. Cite this as: A. Leonardi, A. Di Stefano, L. Motterle, B. Zavan, G. Abatangelo and P. Brun, Clinical & Experimental Allergy, 2011 (41) 52–60.     

Transforming Growth Factor‐β Suppressed Id‐1 Expression in a smad3‐Dependent Manner in LoVo Cells

TGF‐β plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id‐1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF‐β in regulating Id‐1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF‐β on Id‐1 expression were analyzed using a luciferase reporter assay, RT‐PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF‐β1 downregulated Id‐1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF‐β1‐induced 4×SBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF‐β1‐induced 3×AP‐1 luciferase reporter activity. However, the suppression of Id‐1 by TGF‐β1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF‐β1 stimulated cellular proliferation and p21^Waf1 protein expression, which might be mediated by suppressing Id‐1 expression. In conclusion, this study demonstrated that TGF‐β1 suppressed Id‐1 expression in a smad3‐dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF‐β function in colorectal cancer cells. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Elevated TGFβ–Smad signalling in experimental Pkd1 models and human patients with polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited renal disease characterized by many fluid‐filled cysts and interstitial fibrosis in the kidneys, leading to chronic renal failure. During cystogenesis the renal tubules undergo extensive structural alterations that are accompanied by altered cellular signalling, directly and/or indirectly regulated by the PKD1 and PKD2 proteins. Since transforming growth factor (TGF)‐β signalling modulates cell proliferation, differentiation, apoptosis, adhesion and migration of various cell types, we studied the activation of this signalling pathway in Pkd1‐mutant mouse models at different stages of the disease. Therefore, we analysed expression of the TGFβ–Smad signalling pathway and its target genes in different Pkd1 mutant mouse models in various stages of polycystic disease. Nuclear accumulation of P‐Smad2 in cyst lining epithelial cells was not observed in the initiation phase but was observed at mild and more advanced stages of PKD. This coincides with mild fibrosis and increased mRNA levels of TGFβ target genes, such as fibronectin, collagen type I, plasminogen activator inhibitor 1 and matrix metalloproteinase‐2. At this stage many interstitial fibroblasts were found around cysts, which also showed nuclear localization for P‐Smad2. However, bone morphogenetic protein (BMP) signalling, which can antagonize TGFβ signalling, is not affected, since nuclear expression of P‐Smad1/5/8 and expression of the BMP target gene, inhibitor of DNA binding/differential‐1 (ID‐1) is not altered compared to wild‐type controls. Also, human kidneys with progressive ADPKD showed increased nuclear localization of P‐Smad2, while in general expression of P‐Smad1/5/8 was weak. These results exclude TGFβ signalling at the initiation of cystogenesis, but indicate an important role during cyst progression and in fibrogenesis of progressive ADPKD. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pulmonary inflammation in mice with collagen‐induced arthritis is conditioned by complete Freund's adjuvant and regulated by endogenous IFN‐γ

Following immunization with collagen II (CII) in complete Freund's adjuvant (CFA), DBA/1 mice develop arthritis of major joints. This collagen‐induced arthritis (CIA) is used as a model for rheumatoid arthritis (RA) in man. Inflammatory changes in lung tissue commonly occur in RA. However, evidence for pulmonary inflammation in CIA is scarce and ambiguous. Here, we demonstrate pulmonary inflammation accompanying CIA in wild‐type DBA/1 mice. In IFN‐γ receptor‐deficient (IFN‐γR KO) mice, inflammation was more frequent and more severe. Injection of CFA only (without CII) proved to be as efficient in eliciting pulmonary inflammation as immunization with CFA + CII, though being less effective in causing arthritis. Significant correlation in severity between joint and pulmonary involvement could not be demonstrated. Macroscopic, microscopic, and functional characteristics of pulmonary inflammation in the mice resembled those seen in human RA. Increased inflammation in IFN‐γR KO mice was accompanied by augmented expression of various cytokines and chemokines, as measured by RT‐PCR on affected tissue. Treatment with a TNF‐α inhibitor ameliorated lung pathology. We conclude that CIA in DBA/1 mice is accompanied by pulmonary inflammation. Although both disease processes are kept in check by endogenous IFN‐γ, lack of strict parallelism indicates that overlap in their pathogeneses is partial.     

Synergistic effect of TGF‐β superfamily members on the induction of Foxp3+ Treg

TGF‐β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF‐β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF‐β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP‐2 and ‐4, are unable to induce non‐regulatory T cells to become Foxp3⁺ Treg. Neutralization studies with Noggin have revealed that BMP‐2/4 and the BMP receptor signaling pathway is not required for TGF‐β to induce naïve CD4⁺CD25^? cells to express Foxp3; however, BMP‐2/4 and TGF‐β have a synergistic effect on the induction of Foxp3⁺ Treg. BMP‐2/4 affects non‐Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ Treg induced by TGF‐β. Data further advocate that TGF‐β is a key signaling factor for Foxp3⁺ Treg development. In addition, the synergistic effect of BMP‐2/4 and TGF‐β indicates that the simultaneous manipulation of TGF‐β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.     

Attenuated TGF‐β1 responsiveness of dendritic cells and their precursors in atopic dermatitis

The responsiveness of DCs and their precursors to transforming growth factor beta1 (TGF‐β1) affects the nature of differentiating DC subsets, which are essential for the severity of atopic dermatitis (AD). To evaluate TGF‐β signaling in monocytes and monocyte‐derived DCs of AD patients compared with that of controls, in vitro generated Langerhans cell (LC) like DCs, expression of TGF‐β receptors, phospho‐Smad2/3 and Smad7 were evaluated. Furthermore, TNF‐α expression and synergistic effects of TNF‐α upon TGF‐β signaling and DC generation were evaluated. We found LC‐like DC differentiation of monocytes from AD patients in response to TGF‐β1 was remarkably reduced and TGF‐β1 receptor expression was significantly lower compared with that of healthy controls. Attenuated TGF‐β1 responsiveness mirrored by lower phospho‐Smad2/3 expression after TGF‐β1 stimulation and higher expression of inhibitory Smad7 was observed in monocytes from AD patients. During DC generation, mRNA expression of Smad7 was relatively higher in LC‐like DCs of AD patients. Lower TNF‐α expression of monocytes from AD patients might further contribute to attenuated TGF‐β signaling in the disease since TNF‐α had synergistic effects on TGF‐β1 signaling and LC generation through mediating the degradation of Smad7. Our results demonstrate alleviated TGF‐β1 signaling together with the amount of soluble co‐factors might direct the nature of differentiating DCs.     

Blockade of interleukin‐6 receptor enhances the anti‐arthritic effect of glucocorticoids without decreasing bone mineral density in mice with collagen‐induced arthritis

In a mouse arthritis model, we investigated whether interleukin‐6 receptor (IL‐6R) blockade would enhance the anti‐arthritic effect of glucocorticoids (GCs). DBA/1J mice were immunized with type II collagen (CII), and were treated with prednisolone (PSL) and/or anti‐mouse IL‐6R antibody (MR16‐1). Also, the effects of IL‐6 on gene expression and the nuclear translocation of glucocorticoid receptors (GRs) were examined in cultured cells treated with dexamethasone (DEX). PSL reduced the arthritis score dose‐dependently in the collagen‐induced arthritis (CIA) mouse model. The arthritis score in the PSL (3 mg/kg) + MR16‐1 group was lower than in the PSL (3 mg/kg) group, and at the same level as in the PSL (6 mg/kg) group. Lumbar vertebra bone mineral density (BMD) was decreased significantly in CIA mice and was higher in the PSL (3 mg/kg) + MR16‐1 group than in the PSL (6 mg/kg) group. In the in‐vitro synovial cells, IL‐6 pretreatment attenuated the inhibitory effect of DEX on cyclooxygenase (COX)‐2 expression and inhibited the nuclear translocation of GR induced by DEX. In contrast, in MC3T3‐E1 osteoblastic cells, IL‐6 pretreatment exacerbated the decrease in expression of osteocalcin and the increase in expression of receptor activator of nuclear factor kappa‐B ligand (RANKL) by DEX. We demonstrated that IL‐6 signalling blockade by an anti‐IL‐6R antibody can augment the anti‐arthritic effect of GCs and inhibit the bone loss they cause.     

Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse‐type gastric carcinoma‐initiating cells is inhibited by TGF‐β

Aldehyde dehydrogenase 1 (ALDH1) has been shown to serve as a marker for cancer‐initiating cells (CICs), but little is known about the regulation of the CIC functions of ALDH1+ cancer cells. We isolated ALDH1+ cells from human diffuse‐type gastric carcinoma cells and characterized these cells using an Aldefluor assay. ALDH1+ cells constituted 5–8% of the human diffuse‐type gastric carcinoma cells, OCUM‐2MLN and HSC‐39; were more tumourigenic than ALDH1? cells; and were able to self‐renew and generate heterogeneous cell populations. Using gene expression microarray analyses, we identified REG4 (regenerating islet‐derived family, member 4) as one of the genes up‐regulated in ALDH1+ cells, and thus as a novel marker for ALDH1+ tumour cells. Induced expression of REG4 enhanced the colony‐forming ability of OCUM‐2MLN cells, while knockdown of REG4 inhibited the tumourigenic potential of ALDH1+ cells. We further found that TGF‐β signalling reduces the expression of ALDH1 and REG4, and the size of the ALDH1+ cell population. In human diffuse‐type gastric carcinoma tissues, the expression of ALDH1 and REG4 correlated with each other, as assessed by immunohistochemistry, and ALDH1 expression correlated inversely with Smad3 phosphorylation as a measure of TGF‐β signalling. These findings illustrate that, in diffuse‐type gastric carcinoma, REG4 is up‐regulated in ALDH1+ CICs, and that the increased tumourigenic ability of ALDH1+ cells depends on REG4. Moreover, TGF‐β down‐regulates ALDH1 and REG4 expression, which correlates with a reduction in CIC population size and tumourigenicity. Targeting REG4 in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse‐type gastric carcinoma. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Role of T cell TGF‐β signaling in intestinal cytokine responses and helminthic immune modulation

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL‐10 or TGF‐β, that are important in suppressing colitis. Helminths induce mucosal T cell IL‐10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL‐10‐dependent manner in WT mice. Helminths also stimulate mucosal TGF‐β release. As TGF‐β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF‐β signaling in helminthic modulation of intestinal immunity. T cell TGF‐β signaling is interrupted in TGF‐β receptor II dominant negative (TGF‐βRII DN) mice by T‐cell‐specific over‐expression of a TGF‐βRII DN. We studied LPMC responses in WT and TGF‐βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF‐β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL‐10 secretion requires intact T cell TGF‐β‐signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF‐βRII DN mice. Thus, T cell TGF‐β signaling is essential for helminthic stimulation of mucosal IL‐10 production, helminthic modulation of intestinal IFN‐γ generation and H. polygyrus‐mediated suppression of chronic colitis.     

Berbamine Enhances the Antineoplastic Activity of Gemcitabine in Pancreatic Cancer Cells by Activating Transforming Growth Factor‐β/Smad Signaling

Drug‐resistance to gemcitabine chemotherapy in pancreatic cancer is still an unsolved problem. Combinations of other chemotherapy drugs with gemcitabine have been shown to increase the efficacy of gemcitabine‐based treatment. In this study, the effect of berbamine on the antitumor activity of gemcitabine was evaluated in human pancreatic cancer cell lines Bxpc‐3 and Panc‐1, and the underlying mechanisms were explored. Our results demonstrated that berbamine exhibited a time‐ and dose‐dependent inhibitory effect in the pancreatic cancer cell lines. Berbamine enhanced gemcitabine‐induced cell growth inhibition and apoptosis in these cells. Combined treatment of berbamine and gemcitabine resulted in down‐regulation of anti‐apoptotic proteins (Bcl‐2, Bcl‐xL) and up‐regulation of pro‐apoptotic proteins (Bax, Bid). More importantly, berbamine treatment in combination with gemcitabine activated the transforming growth factor‐β/Smad (TGF‐β/Smad) signaling pathway, as a result of a decrease in Smad7 and an increase in transforming growth factor‐β receptor II (TβRII) expression. Changes in downstream targets of Smad7, such as up‐regulation of p21 and down‐regulation of c‐Myc and Cyclin D1 were also observed. Therefore, berbamine could enhance the antitumor activity of gemcitabine by inhibiting cell growth and inducing apoptosis, possibly through the regulation of the expression of apoptosis‐related proteins and the activation of TGF‐β/Smad signaling pathway. Our study indicates that berbamine may be a promising candidate to be used in combination with gemcitabine for pancreatic cancer treatment. Anat Rec, 297:802–809, 2014. © 2014 Wiley Periodicals, Inc.     

Distinct expression of interleukin (IL)‐36α, β and γ, their antagonist IL‐36Ra and IL‐38 in psoriasis,rheumatoid arthritis and Crohn's disease

Interleukin (IL)‐36α, IL‐36β and IL‐36γ are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL‐36Ra or IL‐38, another potential IL‐36 inhibitor, limit uncontrolled inflammation. The expression and role of IL‐36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod‐induced mouse skin inflammation and in human psoriasis, expression of IL‐36α, γ and IL‐36Ra, but not IL‐36β and IL‐38 mRNA, was induced and correlated with IL‐1β and T helper type 17 (Th17) cytokines (IL‐17A, IL‐22, IL‐23, CCL20). In mice with collagen‐induced arthritis and in the synovium of patients with RA, IL‐36α, β, γ, IL‐36Ra and IL‐38 were all elevated and correlated with IL‐1β, CCL3, CCL4 and macrophage colony‐stimulating factor (M‐CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium‐induced colitis and in patients with CD, only IL‐36α, γ and IL‐38 were induced at relatively low levels and correlated with IL‐1β and IL‐17A. We suggest that only a minor subgroup of patients with RA (17–29%) or CD (25%) had an elevated IL‐36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL‐36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68⁺ macrophages, dendritic/Langerhans cells and CD79α⁺ plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL‐36β and IL‐36Ra were produced constitutively, but IL‐36α, γ and IL‐38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL‐36 agonists/antagonists ratio.     

B‐cell epitope spreading and inflammation in a mouse model of arthritis is associated with a deficiency in reactive oxygen species production

Autoantibody‐mediated inflammation contributes to the development of rheumatoid arthritis (RA), and anti‐type II collagen (CII) antibodies are present in the serum, synovial fluid, and cartilage of RA patients. We had previously generated and characterized knock‐in mice expressing a germline‐encoded, CII‐specific IgH (B10Q.ACB), which demonstrated positive selection of self‐reactive B cells. Here, we show that despite the spontaneous production of CII‐specific autoantibodies, B10Q.ACB mice are protected from collagen‐induced arthritis. Introducing a mutation in the Ncf1 gene, leading to ROS deficiency, breaks this strong arthritis resistance. Disease development in Ncf1‐mutated B10Q.ACB mice is associated with an enhanced germinal center formation but without somatic mutations of the auto‐reactive B cells, increased T‐cell responses and intramolecular epitope‐spreading. Thus, ROS‐mediated B‐cell tolerance to a self‐antigen could operate by limiting the expansion of the auto‐reactive B‐cell repertoire, which has important implications for the understanding of epitope spreading phenomena in rheumatoid arthritis and other autoimmune diseases.     

Tissue transglutaminase enhances collagen type II‐induced arthritis and modifies the immunodominant T‐cell epitope CII260‐270

The calcium‐dependent enzyme tissue transglutaminase (tTG) is associated with diverse biological functions, such as induction of apoptosis, modeling of the extracellular matrix, receptor‐mediated endocytosis, cell growth and differentiation, cell adhesion and signal transduction. Also, it may deamidate glutamine residues to glutamic acid and catalyze cross‐linking of proteins. In this study, we have investigated the impact of tTG for posttranslational modifications and cross‐linking of the immunodominant T‐cell epitope CII260‐270 and their effects on the collagen‐induced arthritis, an animal model for rheumatoid arthritis. By using mass spectrometry analysis and hybridoma assays, we have demonstrated that tTG could perform both types of modifications (deamidation and cross‐link formation) on the immunodominant T‐cell epitope CII259‐273. Replacement of the glutamine at position 267 with glutamic acid leads to a decreased binding affinity to MHC II. T cells recognized both non‐modfied (Q²⁶⁷) and modified (E²⁶⁷) CII259‐273‐peptides. We also show that administration of tTG leads to increased incidence, severity and histopathological manifestations of collagen‐induced arthritis in mice. Moreover, we conclude that both processes, deamidation and cross‐linking, are involved in the tTG‐catalyzed reactions, and in vivo administration of tTG enhances arthritis severity and joint destruction in mice.     

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis,selectively induces pro‐inflammatory cytokines and IL‐10 and is regulated by TNF‐α, IL‐10 and IFN‐γ

Leukocyte immunoglobulin‐like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane‐bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross‐linking of LILRA5 on monocytes induced production of pro‐inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common γ chain of the FcR and LILRA5 cross‐linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro‐inflammatory cytokines as well as IL‐10. LILRA5 mRNA and protein expression was tightly regulated by TNF‐α, IL‐10 and IFN‐γ. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.