Annunciata Vecchi

Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response

Pentraxins are a superfamily of conserved proteins that are characterized by a cyclic multimeric structure. The classical short pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are acute-phase proteins produced in the liver in response to inflammatory mediators. Short pentraxins regulate innate resistance to microbes and the scavenging of cellular debris and extracellular matrix components. In contrast, long pentraxins have an unrelated, long amino-terminal domain coupled to the carboxy-terminal pentraxin domain, and differ, with respect to short pentraxins, in their gene organization, chromosomal localization, cellular source, and in their stimuli-inducing and ligand-recognition ability. To investigate the in vivo function of the long pentraxin PTX3, we generated mice deficient in Ptx3 by homologous recombination. Ptx3-null mice were susceptible to invasive pulmonary aspergillosis. Ptx3 binds selected microbial agents, including conidia of Aspergillus fumigatus, and we found that susceptibility of Ptx3-null mice was associated with defective recognition of conidia by alveolar macrophages and dendritic cells, as well as inappropriate induction of an adaptive type 2 response. Thus, the long pentraxin Ptx3 is a secreted pattern-recognition receptor that has a non-redundant role in resistance to selected microbial agents, in particular to the opportunistic fungal pathogen Aspergillus fumigatus.

Complement Activation Determines the Therapeutic Activity of Rituximab In Vivo

Rituximab is an anti-CD20 chimeric mAb effective for the treatment of B-NHL. It can lyse lymphoma cells in vitro through both C- and Ab-dependent cellular cytotoxicity. The mechanism of action of rituximab in vivo is however still unclear. We have set up a new in vivo model in nonimmunodeficient mice by stable transduction of the human CD20 cDNA in the murine lymphoma line EL4. Animals injected i.v. with the EL4-CD20+ lymphoma cells died within 30 days with evident liver, spleen, and bone marrow involvement, confirmed by immunohistochemistry and PCR analysis. A single injection of rituximab or the murine anti-CD20 Ab 1F5, given i.p. 1 day after the tumor, cured 100% of the animals. Indeed, at week 4 after tumor cell inoculation, CD20+ cells were undetectable in all organs analyzed in rituximab-treated animals, as determined by immunohistochemistry and PCR. Rituximab had no direct effect on tumor growth in vitro. Depletion of either NK cells or neutrophils or both in tumor-injected animals did not affect the therapeutic activity of the drug. Similarly, rituximab was able to eradicate tumor cells in athymic nude mice, suggesting that its activity is T cell independent. In contrast, the protective activity of rituximab or the 1F5 Ab was completely abolished in syngeneic knockout animals lacking C1q, the first component of the classical pathway of C (C1qa−/−). These data demonstrate that C activation is fundamental for rituximab therapeutic activity in vivo.

The Contribution of the Toll-Like/IL-1 Receptor Superfamily to Innate and Adaptive Immunity to Fungal Pathogens In Vivo

In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.

Autocrine Production of IL-10 Mediates Defective IL-12 Production and NF-κB Activation in Tumor-Associated Macrophages

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 production was associated with lack of p50/p65 NF-κB activation. TAM produced increased amounts of the immunosuppressive cytokine IL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growth an IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and results in the promotion of the IL-10+ IL-12− phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines present in the tumor microenvironment, such as TGF-β, may complement therapeutic strategies aimed at activating type I antitumor immune responses.

The pattern of response to anti-interleukin-1 treatment distinguishes two subsets of patients with systemic-onset juvenile idiopathic arthritis

OBJECTIVE To assess the clinical response to interleukin-1 (IL-1) blockade and in vitro IL-1beta and IL-18 secretion in patients with systemic-onset juvenile idiopathic arthritis (JIA). METHODS Twenty-two patients with systemic-onset JIA were treated with the IL-1 receptor antagonist (IL-1Ra) anakinra. Monocytes from 18 patients and 20 healthy donors were activated by different Toll-like receptor ligands. Intracellular and secreted IL-1beta and IL-18 were analyzed by Western blotting and enzyme-linked immunosorbent assay. RESULTS Ten patients with systemic-onset JIA exhibited a dramatic response to anakinra and were classified as complete responders. Eleven patients had an incomplete response or no response, and 1 patient could not be classified in terms of response. Compared with patients who had an incomplete response or no response, complete responders had a lower number of active joints (P = 0.02) and an increased absolute neutrophil count (P = 0.02). In vitro IL-1beta and IL-18 secretion in response to various stimuli was not increased and was independent of treatment efficacy. Likewise, secretion of IL-1Ra by monocytes from patients with systemic-onset JIA was not impaired. An overall low level of IL-1beta secretion upon exposure to exogenous ATP was observed, unrelated to treatment responsiveness or disease activity. CONCLUSION Two subsets of systemic-onset JIA can be identified according to patient response to IL-1 blockade. The 2 subsets appear to be characterized by some distinct clinical features. In vitro secretion of IL-1beta and IL-18 by monocytes from patients with systemic-onset JIA is not increased and is independent of both treatment outcome and disease activity.

Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines

The canonical concept of a receptor includes specific ligand recognition, usually with high affinity and specificity, and signaling. Decoy receptors recognize certain inflammatory cytokines with high affinity and specificity, but are structurally incapable of signaling or presenting the agonist to signaling receptor complexes. They act as a molecular trap for the agonist and for signaling receptor components. The interleukin-1 type II receptor (IL-1RII) was the first pure decoy to be identified. Decoy receptors have subsequently been identified for members of the tumor necrosis factor receptor and IL-1R families. Moreover, silent nonsignaling receptors could act as decoys for chemokines. Therefore, the use of decoy receptors is a general strategy to regulate the action of primary pro-inflammatory cytokines and chemokines.

Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses

Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-gamma, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-gamma. TNF alone or combined with IFN-gamma also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelmans disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.

Inhibition of monocyte chemotactic protein-1 synthesis by statins

The beneficial effects of statins on the reduction of cardiovascular events has been partly attributed to their anti-inflammatory properties. In the complex of the different pathogenetic events leading to atherosclerosis, recent data suggest a central role of monocyte chemotactic protein-1 (MCP-1), because mice knock-out for MCP-1 or its receptor CC-chemokine receptor 2 were considerably resistant to plaque formation. In this study we investigated the effect of different statins on in vitro and in vivo production of MCP-1. Lovastatin and simvastatin caused a dose-dependent inhibition of MCP-1 production in peripheral blood mononuclear cells exposed to lipopolysaccharide or inactivated Streptococcus hemoliticus and in human endothelial cells exposed to interleukin-1β. The addition of mevalonate overrode the inhibitory effect of statins indicating that mevalonate-derived products are important for chemokine production. The in vivo anti-inflammatory effect of statins was investigated using the mouse air-pouch model of local inflammation. Lovastatin and pravastatin were orally administered to mice according to a treatment schedule that significantly inhibited the hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity without affecting total blood cholesterol. At the dose of 10 mg/kg, lovastatin and pravastatin reduced by approximately 50% the lipopolysaccharide-induced leukocytes recruitment and the exudate MCP-1 production. In conclusion, statins, by inhibiting mevalonate-derived products, reduced both in vitro and in vivo the production of chemokines involved in leukocyte migration, and this effect is unrelated to their cholesterol-lowering action.

Increased Survival, Proliferation, and Migration in Metastatic Human Pancreatic Tumor Cells Expressing Functional CXCR4

In this study, we have evaluated 11 pancreatic tumor cell lines and tumor cells from surgical samples of patients with pancreatic adenocarcinoma for expression of the chemokine receptor CXCR4. Six of 11 cell lines expressed detectable mRNA of CXCR4, with three cell lines (AsPC1, Capan1, and Hs766T) having substantial amounts of transcripts. Expression was higher in lines derived from metastatic lesions compared with those derived from primary tumors. Different inflammatory cytokines did not modify expression, whereas IFN-γ down-regulated and hypoxia up-regulated CXCR4 transcripts. Transcript expression was associated with surface expression in pancreatic carcinoma cell lines. All surgical carcinoma samples tested expressed higher levels of CXCR4 than normal pancreatic ducts, which were used as reference tissue. The chemokine CXCL12 induced chemotaxis in CXCR4-positive pancreatic carcinoma cell lines, which was inhibited by anti-CXCR4 monoclonal antibody and by the antagonist AMD3100. Transendothelial migration, Matrigel invasion, and activation of matrix metalloproteases were also enhanced by CXCL12. In CXCR4-positive cell lines, CXCL12 stimulated cell proliferation. The cell line Hs766T produces high levels of CXCL12, and addition of the CXCR4 antagonist AMD3100 partially inhibited proliferation, indicating an autocrine loop. Moreover, the addition of exogenous CXCL12 inhibited apoptosis induced by serum starvation. These results indicate that the CXCR4 receptor is frequently expressed in metastatic pancreatic tumor cells. CXCR4 not only stimulates cell motility and invasion but also promotes survival and proliferation. Strategies to target CXCR4 expressed on tumor cells may be of benefit in patients with pancreatic cancer.

The role of chemokines in the regulation of dendritic cell trafficking

The immunobiology of dendritic cells (DC) involves localization in tissues and trafficking via the lymph or blood to lymphoid organs. Appropriate assays representative of different steps of DC trafficking (e.g., reverse transmigration) provide the tools to dissect the migratory properties of these cells. Chemokines have emerged as important regulators of DC migration. DC are both the target and the source of chemokines. DC express receptors for and respond to a set of chemoattractants that overlap with, but are distinct from, those active on other leukocytes. Differential expression of the CCR6 receptor reveals heterogeneity among DC populations. Functional maturation is associated with loss of responsiveness to chemokines present at sites of inflammation and acquisition of a receptor repertoire that renders these cells responsive to signals that guide their localization in lymphoid organs. A better understanding of the molecular basis of DC trafficking may provide molecular and conceptual tools to direct and modulate DC traffic as a strategy to up‐regulate and orient specific immunity. J. Leukoc. Biol. 66: 1–9; 1999.