Mast cells are associated with the onset and progression of celiac disease.

BACKGROUND: Celiac disease (CD) is an immune-mediated disorder characterized by an accumulation of immune cells in the duodenal mucosa as a consequence of both adaptive and innate immune responses to undigested gliadin peptides. Mast cells (MCs) are innate immune cells that are a major source of costimulatory signals and inflammatory mediators in the intestinal mucosa. Although MCs have previously been associated with CD, functional studies have never been performed. OBJECTIVE: We aimed at evaluating the role of MCs in the pathogenesis of CD. METHODS: Intestinal biopsy specimens of patients with CD were scored according to the Marsh classification and characterized for leukocyte infiltration and MC distribution. Moreover, MC reactivity to gliadin and its peptides was characterized by using in vitro assays. RESULTS: Infiltrating MCs were associated with the severity of mucosal damage, and their numbers were increased in patients with higher Marsh scores. MCs were found to directly respond to nonimmunodominant gliadin fragments by releasing proinflammatory mediators. Immunohistochemical characterization of infiltrating MCs and the effects of gliadin peptides on intestinal MCs indicated an increase in proinflammatory MC function in advanced stages of the disease. This was also associated with increased neutrophil accumulation, the prevalence of M1 macrophages, and the severity of tissue damage. CONCLUSION: We provide a description of the progressive stages of CD, in which MCs are the hallmark of the inflammatory process. Thus the view of CD should be revised, and the contribution of MCs in the onset and progression of CD should be reconsidered in developing new therapeutic approaches.

Mast cells contribute to autoimmune diabetes by releasing interleukin-6 and failing to acquire a tolerogenic IL-10+ phenotype.

Mast cells (MCs) are innate immune cells that exert positive and negative immune modulatory functions capable to enhance or limit the intensity and/or duration of adaptive immune responses. Although MCs are crucial to regulate T cell immunity, their action in the pathogenesis of autoimmune diseases is still debated. Here we demonstrate that MCs play a crucial role in T1D pathogenesis so that their selective depletion in conditional MC knockout NOD mice protects them from the disease. MCs of diabetic NOD mice are overly inflammatory and secrete large amounts of IL-6 that favors differentiation of IL-17-secreting T cells at the site of autoimmunity. Moreover, while MCs of control mice acquire an IL-10+ phenotype upon interaction with FoxP3+ Treg cells, MCs of NOD mice do not undergo this tolerogenic differentiation. Our data indicate that overly inflammatory MCs unable to acquire a tolerogenic IL-10+ phenotype contribute to the pathogenesis of autoimmune T1D.

C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation.

Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa(-/-)) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa(-/-) mice. Bone marrow (BM) chimeras between C1qa(-/-) and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth.

Interleukin-17A promotes the growth of human germinal center derived non-Hodgkin B cell lymphoma.

Interleukin (IL)-17A belongs to IL-17 superfamily and binds the heterodimeric IL-17 receptor (R)(IL-17RA/IL-17RC). IL-17A promotes germinal center (GC) formation in mouse models of autoimmune or infectious diseases, but the role of IL-17A/IL-17AR complex in human neoplastic GC is unknown. In this study, we investigated expression and function of IL-17A/IL-17AR in the microenvironments of 44 B cell non-Hodgkin lymphomas (B-NHL) of GC origin (15 follicular lymphomas, 17 diffuse large B cells lymphomas and 12 Burkitt lymphomas) and 12 human tonsil GC. Furthermore, we investigated the role of IL-17A in two in vivo models of GC B cell lymphoma, generated by s.c. injection of SU-DHL-4 and OCI-Ly8 cell lines in Severe combined immunodeficiency (SCID)/Non Obese Diabetic (NOD) mice. We found that: (i) B-NHL cell fractions and tonsil GC B cells expressed IL-17RA/IL-17RC, (ii) IL-17A signaled in both cell types through NF-kBp65, but not p38, ERK-1/2, Akt or NF-kBp50/105, phosphorylation, (iii) IL-17A was expressed in T cells and mast cells from neoplastic and normal GC microenvironments, (iv) IL-17A rendered tonsil GC B cells competent to migrate to CXCL12 and CXCL13 by downregulating RGS16 expression; (v) IL-17A stimulated in vitro proliferation of primary B-NHL cells; (vi) IL-17A (1 µg/mouse-per dose) stimulated B-NHL growth in two in vivo models by enhancing tumor cell proliferation and neo-angiogenesis. This latter effect depended on IL-17A-mediated induction of pro-angiogenic gene expression in tumor cells and direct stimulation of endothelial cells. These data define a previously unrecognized role of human IL-17A in promoting growth of GC-derived B-NHL and modulating normal GC B cell trafficking.

Mast Cells Infiltrating Inflamed or Transformed Gut Alternatively Sustain Mucosal Healing or Tumor Growth.

Mast cells (MC) are immune cells located next to the intestinal epithelium with regulatory function in maintaining the homeostasis of the mucosal barrier. We have investigated MC activities in colon inflammation and cancer in mice either wild-type (WT) or MC-deficient (Kit(W-sh)) reconstituted or not with bone marrow-derived MCs. Colitis was chemically induced with dextran sodium sulfate (DSS). Tumors were induced by administering azoxymethane (AOM) intraperitoneally before DSS. Following DSS withdrawal, Kit(W-sh) mice showed reduced weight gain and impaired tissue repair compared with their WT littermates or Kit(W-sh) mice reconstituted with bone marrow-derived MCs. MCs were localized in areas of mucosal healing rather than damaged areas where they degraded IL33, an alarmin released by epithelial cells during tissue damage. Kit(W-sh) mice reconstituted with MC deficient for mouse mast cell protease 4 did not restore normal mucosal healing or reduce efficiently inflammation after DSS withdrawal. In contrast with MCs recruited during inflammation-associated wound healing, MCs adjacent to transformed epithelial cells acquired a protumorigenic profile. In AOM- and DSS-treated WT mice, high MC density correlated with high-grade carcinomas. In similarly treated Kit(W-sh) mice, tumors were less extended and displayed lower histologic grade. Our results indicate that the interaction of MCs with epithelial cells is dependent on the inflammatory stage, and on the activation of the tissue repair program. Selective targeting of MCs for prevention or treatment of inflammation-associated colon cancer should be timely pondered to allow tissue repair at premalignant stages or to reduce aggressiveness at the tumor stage.

SOCS2 Controls Proliferation and Stemness of Hematopoietic Cells under Stress Conditions and Its Deregulation Marks Unfavorable Acute Leukemias.

Hematopoietic stem cells (HSC) promptly adapt hematopoiesis to stress conditions, such as infection and cancer, replenishing bone marrow-derived circulating populations, while preserving the stem cell reservoir. SOCS2, a feedback inhibitor of JAK-STAT pathways, is expressed in most primitive HSC and is upregulated in response to STAT5-inducing cytokines. We demonstrate that Socs2 deficiency unleashes HSC proliferation in vitro, sustaining STAT5 phosphorylation in response to IL3, thrombopoietin, and GM-CSF. In vivo, SOCS2 deficiency leads to unrestricted myelopoietic response to 5-fluorouracil (5-FU) and, in turn, induces exhaustion of long-term HSC function along serial bone marrow transplantations. The emerging role of SOCS2 in HSC under stress conditions prompted the investigation of malignant hematopoiesis. High levels of SOCS2 characterize unfavorable subsets of acute myeloid and lymphoblastic leukemias, such as those with MLL and BCR/ABL abnormalities, and correlate with the enrichment of genes belonging to hematopoietic and leukemic stemness signatures. In this setting, SOCS2 and its correlated genes are part of regulatory networks fronted by IKZF1/Ikaros and MEF2C, two transcriptional regulators involved in normal and leukemic hematopoiesis that have never been linked to SOCS2. Accordingly, a comparison of murine wt and Socs2(-/-) HSC gene expression in response to 5-FU revealed a significant overlap with the molecular programs that correlate with SOCS2 expression in leukemias, particularly with the oncogenic pathways and with the IKZF1/Ikaros and MEF2C-predicted targets. Lentiviral gene transduction of murine hematopoietic precursors with Mef2c, but not with Ikzf1, induces Socs2 upregulation, unveiling a direct control exerted by Mef2c over Socs2 expression.

HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma.

We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10(4) cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

The hepatic expression of vitamin D receptor is inversely associated with the severity of liver damage in genotype 1 chronic hepatitis C patients.

BACKGROUND/AIMS: Low 25-hydroxyvitamin D serum levels have been associated with the severity of liver fibrosis in genotype 1 chronic hepatitis C patients (G1CHC), and experimental evidence suggested a hepatoprotective role of vitamin D via interaction with hepatic vitamin D receptor (VDR). We assessed the hepatic expression of VDR protein and its association with liver disease severity. METHODS: Ninety-one consecutive patients with biopsy-proven G1CHC and available frozen liver tissue were evaluated. Ten subjects without chronic liver diseases and nine patients with autoimmune hepatitis served as controls. The hepatic expression of VDR protein was assessed by Western blot for quantification and by immunohistochemistry for morphological distribution. RESULTS: Liver VDR protein was mainly localized in hepatocytes and cholangiocytes, and its expression by a Western blot was similar in chronic hepatitis C (CHC) and controls (1.83 ± 0.97 vs 2.18 ± 0.62, P = .14) but was lower in autoimmune hepatitis (0.84 ± 0.14, P < .001). The expression was lower in CHC with severe necroinflammatory activity (1.44 ± 0.87) vs both controls and CHC with grade 1-2 inflammation (1.94 ± 0.97, P = .01 and P = .03, respectively) but higher compared with autoimmune hepatitis (P = .007). A similar difference was observed in CHC patients with F3-F4 fibrosis whose VDR expression (1.51 ± 1.07) was also lower compared with controls and CHC with F0-F2 fibrosis (1.98 ± 0.89, P = .02 and P = .04, respectively) but higher vs autoimmune hepatitis (P = .003). At multivariate logistic regression analysis, low VDR protein expression remained associated with severe necroinflammatory activity and severe fibrosis (odds ratio 0.543,95% confidence interval 0.288-0.989, P = .04; and odds ratio 0.484,95% confidence interval 0.268-0.877, P = .01, respectively) in CHC after correction for clinical, biochemical, and histological features. CONCLUSION: In a cohort of G1CHC patients, the hepatic expression of VDR protein is associated with the severity of both liver fibrosis and inflammation.

Mast cells boost myeloid-derived suppressor cell activity and contribute to the development of tumor-favoring microenvironment.

Inflammation plays crucial roles at different stages of tumor development and may lead to the failure of immune surveillance and immunotherapy. Myeloid-derived suppressor cells (MDSC) are one of the major components of the immune-suppressive network that favors tumor growth, and their interaction with mast cells is emerging as critical for the outcome of the tumor-associated immune response. Herein, we showed the occurrence of cell-to-cell interactions between MDSCs and mast cells in the mucosa of patients with colon carcinoma and in the colon and spleen of tumor-bearing mice. Furthermore, we demonstrated that the CT-26 colon cancer cells induced the accumulation of CD11b(+)Gr1(+) immature MDSCs and the recruitment of protumoral mast cells at the tumor site. Using ex vivo analyses, we showed that mast cells have the ability to increase the suppressive properties of spleen-derived monocytic MDSCs, through a mechanism involving IFNγ and nitric oxide production. In addition, we demonstrated that the CD40:CD40L cross-talk between the two cell populations is responsible for the instauration of a proinflammatory microenvironment and for the increase in the production of mediators that can further support MDSC mobilization and tumor growth. In light of these results, interfering with the MDSC:mast cell axis could be a promising approach to abrogate MDSC-related immune suppression and to improve the antitumor immune response.

Mast cells control the expansion and differentiation of IL-10-competent B cells.

The discovery of B cell subsets with regulatory properties, dependent on IL-10 production, has expanded our view on the mechanisms that control inflammation. Regulatory B cells acquire the ability to produce IL-10 in a stepwise process: first, they become IL-10 competent, a poised state in which B cells are sensitive to trigger signals but do not actually express the Il-10 gene; then, when exposed to appropriate stimuli, they start producing IL-10. Even if the existence of IL-10-competent B cells is now well established, it is not yet known how different immune cell types cross talk with B cells and affect IL-10-competent B cell differentiation and expansion. Mast cells (MCs) contribute to the differentiation and influence the effector functions of various immune cells, including B lymphocytes. In this study, we explored whether MCs could play a role in the expansion of IL-10-competent B cells and addressed the in vivo relevance of MC deficiency on the generation of these cells. We show that MCs can expand IL-10-competent B cells, but they do not directly induce IL-10 production; moreover, the absence of MCs negatively affects IL-10-competent B cell differentiation. Noteworthy, our findings reveal that the CD40L/CD40 axis plays a significant role in MC-driven expansion of IL-10-competent B cells in vitro and highlight the importance of MC CD40L signaling in the colon.

Stromal niche communalities underscore the contribution of the matricellular protein SPARC to B-cell development and lymphoid malignancies.

Neoplastic B-cell clones commonly arise within secondary lymphoid organs (SLO). However, during disease progression, lymphomatous cells may also colonize the bone marrow (BM), where they localize within specialized stromal niches, namely the osteoblastic and the vascular niche, according to their germinal center- or extra-follicular-derivation, respectively. We hypothesized the existence of common stromal motifs in BM and SLO B-cell lymphoid niches involved in licensing normal B-cell development as well as in fostering transformed B lymphoid cells. Thus, we tested the expression of prototypical mesenchymal stromal cell (MSC) markers and regulatory matricellular proteins in human BM and SLO under physiologically unperturbed conditions and during B-cell lymphoma occurrence. We identified common stromal features in the BM osteoblastic niche and SLO germinal center (GC) microenvironments, traits that were also enriched within BM infiltrates of GC-associated B-cell lymphomas, suggesting that stromal programs involved in central and peripheral B-cell lymphopoiesis are also involved in malignant B-cell nurturing. Among factors co-expressed by stromal elements within these different specialized niches, we identified the pleiotropic matricellular protein secreted protein acidic and rich in cysteine (SPARC). The actual role of stromal SPARC in normal B-cell lymphopoiesis, investigated in Sparc-/- mice and BM chimeras retaining the Sparc-/- genotype in host stroma, demonstrated defective BM and splenic B-cell lymphopoiesis. Moreover, in the Trp53 knockout (KO) lymphoma model, p53-/-/Sparc-/- double-KO mice displayed impaired spontaneous splenic B-cell lymphomagenesis and reduced neoplastic clone BM infiltration in comparison with their p53-/-/Sparc+/+ counterparts. Our results are among the first to demonstrate the existence of common stromal programs regulating both the BM osteoblastic niche and the SLO GC lymphopoietic functions potentially fostering the genesis and progression of B-cell malignancies.

PDGFRß and FGFR2 mediate endothelial cell differentiation capability of triple negative breast carcinoma cells.

Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes. In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation. Vascular-like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular-like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA-mediating silencing, allowed the identification of PDGFRß and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo. In summary, we demonstrated that TNBCs have the ability to form vascular-like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRß and FGFR2-mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.

C1q as a unique player in angiogenesis with therapeutic implication in wound healing.

We have previously shown that C1q is expressed on endothelial cells (ECs) of newly formed decidual tissue. Here we demonstrate that C1q is deposited in wound-healing skin in the absence of C4 and C3 and that C1q mRNA is locally expressed as revealed by real-time PCR and in situ hybridization. C1q was found to induce permeability of the EC monolayer, to stimulate EC proliferation and migration, and to promote tube formation and sprouting of new vessels in a rat aortic ring assay. Using a murine model of wound healing we observed that vessel formation was defective in C1qa(-/-) mice and was restored to normal after local application of C1q. The mean vessel density of wound-healing tissue and the healed wound area were significantly increased in C1q-treated rats. On the basis of these results we suggest that C1q may represent a valuable therapeutic agent that can be used to treat chronic ulcers or other pathological conditions in which angiogenesis is impaired, such as myocardial ischemia.

Pleiotropic antitumor effects of the pan-HDAC inhibitor ITF2357 against c-Myc-overexpressing human B-cell non-Hodgkin lymphomas.

Histone deacetylases (HDAC) extensively contribute to the c-Myc oncogenic program, pointing to their inhibition as an effective strategy against c-Myc-overexpressing cancers. We, thus, studied the therapeutic activity of the new-generation pan-HDAC inhibitor ITF2357 (Givinostat®) against c-Myc-overexpressing human B-cell non-Hodgkin lymphomas (B-NHLs). ITF2357 anti-proliferative and pro-apoptotic effects were analyzed in B-NHL cell lines with c-Myc translocations (Namalwa, Raji and DOHH-2), stabilizing mutations (Raji) or post-transcriptional alterations (SU-DHL-4) in relationship to c-Myc modulation. ITF2357 significantly delayed the in vitro growth of all B-NHL cell lines by inducing G1 cell-cycle arrest, eventually followed by cell death. These events correlated with the extent of c-Myc protein, but not mRNA, downregulation, indicating the involvement of post-transcriptional mechanisms. Accordingly, c-Myc-targeting microRNAs let-7a and miR-26a were induced in all treated lymphomas and the cap-dependent translation machinery components 4E-BP1, eIF4E and eIF4G, as well as their upstream regulators, Akt and PIM kinases, were inhibited in function of the cell sensitivity to ITF2357, and, in turn, c-Myc downregulation. In vivo, ITF2357 significantly hampered the growth of Namalwa and Raji xenografts in immunodeficient mice. Noteworthy, its combination with suboptimal cyclophosphamide, achieved complete remissions in most animals and equaled or even exceeded the activity of optimal cyclophosphamide. Collectively, our findings provide the rationale for testing the clinical advantages of adding ITF2357 to current therapies for the still very ominous c-Myc-overexpressing lymphomas. They equally provide the proof-of-concept for its clinical evaluation in rational combination with the promising inhibitors of B-cell receptor and PI3K/Akt/mTOR axis currently in the process of development.

Challenges and new prospects in hepatosplenic γδ T-cell lymphoma.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoid neoplasms characterized by aggressive clinical behavior and dismal prognosis. Hepatosplenic γδ T-cell lymphoma (γδ-HSTL) is a particular form of PTCL that arises from a small subset of γ/δ T-cell receptor-expressing lymphocytes. γδ-HSTL has a rapidly progressive course and poor outcome due also to its refractoriness to conventional chemotherapy regimens. The very low incidence of γδ-HSTL, along with its propensity to mimic different pathological entities, makes this lymphoma a true diagnostic challenge. In this review, we highlight the biological and clinical features of γδ-HSTL that contribute to making this lymphoma a mostly incurable disease. Moreover, we provide a new insight into the crosstalk between HSTL clones and the bone marrow, liver and spleen vascular microenvironment, in which neoplastic cells reside and proliferate. We further discuss γδ-HSTL associated molecules that might be proposed as potential targets for novel therapeutic approaches.

Bone marrow stroma CD40 expression correlates with inflammatory mast cell infiltration and disease progression in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is a mature B-cell neoplasm characterized by rather indolent clinical course. However, nearly one third of patients experience a rapidly progressive disease with a dismal outcome. Despite the characterization of clone genetics and the recognition of deregulated immunologic stimulation in the pathogenesis of SMZL, little is known about microenvironment dynamics and their potential biological influence on disease outcome. Here we investigate the effect of stroma-intrinsic features on SMZL disease progression by focusing on the microenvironment of the bone marrow (BM), which represents an elective disease localization endorsing diagnostic and prognostic relevance. We show that the quality of the BM stromal meshwork of SMZL infiltrates correlates with time to progression. In particular, we describe the unfavorable prognostic influence of dense CD40 expression by BM stromal cells, which involves the contribution of CD40 ligand (CD40L)-expressing bystander mast cells infiltrating SMZL BM aggregates. The CD40/CD40L-assisted crosstalk between mesenchymal stromal cells and mast cells populating the SMZL microenvironment finds correlation in p53(-/-) mice developing SMZL and contributes to the engendering of detrimental proinflammatory conditions. Our study highlights a dynamic interaction, playing between nonneoplastic elements within the SMZL niche, toward disease progression.

Defective stromal remodeling and neutrophil extracellular traps in lymphoid tissues favor the transition from autoimmunity to lymphoma.

Altered expression of matricellular proteins can become pathogenic in the presence of persistent perturbations in tissue homeostasis. Here, we show that autoimmunity associated with Fas mutation was exacerbated and transitioned to lymphomagenesis in the absence of SPARC (secreted protein acidic rich in cysteine). The absence of SPARC resulted in defective collagen assembly, with uneven compartmentalization of lymphoid and myeloid populations within secondary lymphoid organs (SLO), and faulty delivery of inhibitory signals from the extracellular matrix. These conditions promoted aberrant interactions between neutrophil extracellular traps and CD5(+) B cells, which underwent malignant transformation due to defective apoptosis under the pressure of neutrophil-derived trophic factors and NF-κB activation. Furthermore, this model of defective stromal remodeling during lymphomagenesis correlates with human lymphomas arising in a SPARC-defective environment, which is prototypical of CD5(+) B-cell chronic lymphocytic leukemia (CLL).

IL-34 is overexpressed in the inflamed salivary glands of patients with Sjogren's syndrome and is associated with the local expansion of pro-inflammatory CD14(bright)CD16+ monocytes.

OBJECTIVES: To investigate the expression of IL-34 in labial salivary glands (LSGs) of patients with primary SS (p-SS) and its role in inducing a pro-inflammatory monocyte phenotype. METHODS: LSG biopsies were obtained from 20 patients with p-SS and 10 patients with non-Sjögren's sicca syndrome (n-SS). The expression of IL-34, IL-1ß, TNF-α, IL-17 and IL-23 was assessed by real-time PCR. IL-34 expression was also investigated in LSGs by immunohistochemistry. The frequencies of subpopulations of CD14(+) monocytes were evaluated by flow cytometry among isolated mononuclear cells from peripheral blood and salivary glands from both patients and controls. The role of recombinant IL-34 on isolated peripheral blood mononuclear cells was also evaluated. RESULTS: IL-34 m-RNA was overexpressed in the inflamed salivary glands of p-SS and associated with increased expression of TNF-α, IL-1ß, IL-17 and IL-23p19. The increased expression of IL-34 was confirmed by immunohistochemistry in paraffin-embedded salivary glands from p-SS patients. IL-34 expression was accompanied by the expansion of pro-inflammatory CD14(bright)CD16(+) monocytes in the salivary glands. In vitro stimulation of peripheral blood mononuclear cells with IL-34 induced the expansion of both CD14(+)CD16(-) cells and CD14(bright)CD16(+) cells in p-SS and non-SS subjects. CONCLUSION: IL-34 seems to be involved in the pathogenesis of salivary gland inflammation in p-SS.

The monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation.

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B cells and the extent of infiltration is associated with adverse prognostic factors. We studied blood monocyte population by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate proliferation of T cells in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes cultured with CLL cells were also evaluated. The absolute number of monocytes increased in chronic lymphocytic leukemia patients compared to the number in normal controls (792 ± 86 cells/µL versus 485 ± 46 cells/µL, P=0.003). Higher numbers of non-classical CD14(+)CD16(++) and Tie-2-expressing monocytes were also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment of phagocytosis. We also detected gene alterations such as down-regulation of PTGR2, a reductase able to inactivate prostaglandin E2, indicating immunosuppressive activity. Accordingly, the proliferation of T cells in contact with monocytes from patients was inhibited compared to that of cells in contact with monocytes from normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated "education" of immune elements may also include the establishment of a skewed phenotype in the monocyte/macrophage population.