Carmen Penido

Protective Role of the Inflammatory CCR2/CCL2 Chemokine Pathway through Recruitment of Type 1 Cytotoxic γδ T Lymphocytes to Tumor Beds

Tumor-infiltrating lymphocytes (TILs) are important prognostic factors in cancer progression and key players in cancer immunotherapy. Although γδ T lymphocytes can target a diversity of tumor cell types, their clinical manipulation is hampered by our limited knowledge of the molecular cues that determine γδ T cell migration toward tumors in vivo. In this study we set out to identify the chemotactic signals that orchestrate tumor infiltration by γδ T cells. We have used the preclinical transplantable B16 melanoma model to profile chemokines in tumor lesions and assess their impact on γδ TIL recruitment in vivo. We show that the inflammatory chemokine CCL2 and its receptor CCR2 are necessary for the accumulation of γδ TILs in B16 lesions, where they produce IFN-γ and display potent cytotoxic functions. Moreover, CCL2 directed γδ T cell migration in vitro toward tumor extracts, which was abrogated by anti-CCL2 neutralizing Abs. Strikingly, the lack of γδ TILs in TCRδ-deficient but also in CCR2-deficient mice enhanced tumor growth in vivo, thus revealing an unanticipated protective role for CCR2/CCL2 through the recruitment of γδ T cells. Importantly, we demonstrate that human Vδ1 T cells, but not their Vδ2 counterparts, express CCR2 and migrate to CCL2, whose expression is strongly deregulated in multiple human tumors of diverse origin, such as lung, prostate, liver, or breast cancer. This work identifies a novel protective role for CCL2/CCR2 in the tumor microenvironment, while opening new perspectives for modulation of human Vδ1 T cells in cancer immunotherapy.

Role of Monocyte Chemotactic Protein-1/CC Chemokine Ligand 2 on γδ T Lymphocyte Trafficking during Inflammation Induced by Lipopolysaccharide or Mycobacterium bovis Bacille Calmette-Guérin

γδ T lymphocytes are involved in a great variety of inflammatory and infectious responses. However, the mechanisms by which γδ T lymphocytes migrate to inflamed sites are poorly understood. In this study we investigate the role of monocyte chemotactic protein (MCP)-1 in regulating γδ T cell migration after LPS or Mycobacterium bovis bacille Calmette-Guérin (BCG) challenge. LPS-induced γδ T cell influx was significantly inhibited by either pretreatment with dexamethasone or vaccinia virus Lister 35-kDa chemokine binding protein, vCKBP, a CC chemokine neutralizing protein, suggesting a role for CC chemokines in this phenomenon. LPS stimulation increased the expression of MCP-1 mRNA and protein at the inflammation site within 6 h. It is noteworthy that LPS was unable to increase MCP-1 production or γδ T cell recruitment in C3H/HeJ, indicative of the involvement of Toll-like receptor 4. γδ T cells express MCP-1 receptor CCR2. Pretreatment with anti-MCP-1 mAb drastically inhibited LPS-induced in vivo γδ T cell mobilization. Indeed, MCP-1 knockout mice were unable to recruit γδ T cells to the pleural cavity after LPS stimulation, effect that could be restored by coadministration of MCP-1. In addition, BCG-induced γδ lymphocyte accumulation was significantly reduced in MCP-1 knockout mice when compared with wild-type mice. In conclusion, our results indicate that LPS-induced γδ T lymphocyte migration is dependent on Toll-like receptor 4 and sensitive to both dexamethasone and CC chemokine-binding protein inhibition. Moreover, by using MCP-1 neutralizing Abs and genetically deficient mice we show that LPS- and BCG-induced γδ T lymphocyte influx to the pleural cavity of mice is mainly orchestrated by the CC chemokine MCP-1.

Lipoxin A4 attenuates zymosan-induced arthritis by modulating endothelin-1 and its effects

BACKGROUND AND PURPOSE Lipoxin A4 (LXA4) is a lipid mediator involved in the resolution of inflammation. Increased levels of LXA4 in synovial fluid and enhanced expression of the formyl peptide receptor 2/lipoxin A4 receptor (FPR2/ALX) in the synovial tissues of rheumatoid arthritis patients have been reported. Endothelins (ETs) play a pivotal pro‐inflammatory role in acute articular inflammatory responses. Here, we evaluated the anti‐inflammatory role of LXA4, during the acute phase of zymosan‐induced arthritis, focusing on the modulation of ET‐1 expression and its effects.

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-α, and CXCL-1

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET‐1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan‐induced arthritis. Intra‐articular (i.a.) administration of selective ETA or ETB receptor antagonists (BQ‐123 and BQ‐788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 μg/cavity) markedly reduced knee‐joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ETA or ETB receptor blockade suppressed zymosan‐induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ETA/ETB with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ‐123 or BQ‐788 (i.a.; 15 pmol/cavity) also decreased zymosan‐induced TNF‐α production within 6 h, keratinocyte‐derived chemokine/CXCL1 production within 24 h, and leukotriene B4 at both time‐points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan‐induced inflammation, i.a. injection of ET‐1 (1–30 pmol/cavity) or sarafotoxin S6c (0.1–30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee‐joint synovial tissue expressed ETA and ETB receptors. These findings suggest that endogenous ETs contribute to knee‐joint inflammation, acting through ETA and ETB receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

Synthesis and antimalarial activity of hydroxyethylpiperazine derivatives

The antimalarial activity of hydroxyethylpiperazine derivatives, synthesized from the reaction of (2S,3S)Boc-phenylalanine epoxide with benzylpiperazines in good yields (76-96%), has been evaluated in vitro against the Plasmodium falciparum W2 clone (chloroquine resistant). The results show that some compounds have moderate activity against this parasite and none of the active compounds showed cytotoxicity at high concentration (100 microg/ml).

Leukotriene B4 mediates γδ T lymphocyte migration in response to diverse stimuli

Herein, we investigated the involvement of the 5‐LO‐derived lipid mediator LTB4 in γδ T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB4 triggered γδ T lymphocyte mobilization in vivo, a phenomenon also observed in in vitro chemotaxis assays. The i.pl. injection of Escherichia coli endotoxin (LPS) triggered increased levels of LTB4 in pleural cavities. The in vivo inhibition of LTB4 biosynthesis by the 5‐LO inhibitor zileuton or the FLAP inhibitor MK886 attenuated LPS‐induced γδ T cell accumulation into pleural cavities. Accordingly, 5‐LO KO mice failed to recruit γδ T cells into the inflammatory site after i.pl. LPS. Antagonists of the high‐affinity LTB4 receptor BLT1, CP105,696, and LY292476 also attenuated LPS‐induced γδ T cell accumulation in pleural cavities as well as in vitro chemotaxis toward pleural washes obtained from LPS‐simulated mice. LTB4/BLT1 also accounted for γδ T cell migration induced by i.pl. administration of Mycobacterium bovis BCG or antigen in sensitized mice. BLT1 was expressed on naïve, resident as well as LPS‐recruited γδ T cells. Isolated γδ T cells were found to undergo F‐actin cytoskeleton reorganization when incubated with LTB4 in vitro, confirming that γδ T lymphocytes can respond directly to LTB4. In addition to its direct effect on γδ T cells, LTB4 triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that γδ T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB4/BLT1.

IL-5 accounts for the mouse pleural eosinophil accumulation triggered by antigen but not by LPS.

The involvement of interleukin-5 (IL-5) in the pleural eosinophilia induced by LPS or allergen was investigated. The number of pleural eosinophils in actively sensitized mice increased 24 h after the intrathoracic (i.t.) injection of ovalbumin (12 mg/cavity), peaked within 72 h, and persisted significantly increased for at least 120 h. Despite being less intense, the i.t. injection of LPS (250 ng/cavity) also increased the number of pleural eosinophils at 24 h, returning to basal levels within 72 h. Intraperitoneal pretreatment with monoclonal antibody to IL-5 (TRFK-4 and TRFK-5, 500 mg/kg) suppressed the eosinophil accumulation induced by IL-5 (200 units/cavity) or ovalbumin, but had no effect on the LPS-induced eosinophilia. Transfer of the cell-free pleural washing from LPS-treated donor mice to naive recipient animals led to a selective increase in the eosinophil counts. The co-incubation of the pleural washing from LPS-treated animals with monoclonal antibody to IL-5 failed to modify the phenomenon. The results indicate that IL-5 plays an important role in the antigen-induced accumulation of eosinophils in vivo, but not in the eosinophilia triggered by LPS.

Requirement for lymphocytes and resident macrophages in LPS-induced pleural eosinophil accumulation.

In this study we investigated the involvement of inflammatory cells in the pleural accumulation of eosinophils induced by lipopolysaccharide (LPS). Intrathoracic (i.t.) injection of LPS (250 ng/cavity) into rats induced a significant eosinophil accumulation that developed within 24 h, was maximal at 48 h, and returned to control values within 120 h. This eosinophil influx was preceded by a huge neutrophil influx within 4 h and accompanied by a mononuclear cell accumulation between 24 and 48 h. Pretreatment with an antineutrophil monoclonal antibody (RP‐3, 2 ml per animal) selectively reduced the number of circulating neutrophils within 8 h but failed to alter the LPS‐induced eosinophilia. Similarly, platelet depletion with an anti‐rat platelet antiserum did not alter the LPS‐induced eosinophil accumulation. Cyclosporine (50 mg/kg, 12 and 2 h before) partially inhibited (51%) the LPS‐induced pleural eosinophilia, whereas the eosinophilia was not changed by prior degranulation of pleural mast cells with polymyxin B (10 μg/cavity, 24 h before). Moreover, selective depletion of T lymphocytes using an anti‐Thy 1.0 monoclonal antibody significantly inhibited the eosinophilia triggered by LPS. The i.t. injection of liposomes containing dichloromethylene diphosphonate significantly reduced (65%) the number of resident macrophages after 5 days. Under this condition, the eosinophil infiltration induced by LPS was completely inhibited. Accordingly, the i.t. injection of supernatant from macrophage monolayers, obtained from the pleural cavities of LPS‐injected rats, into naive recipient animals led to a twofold increase in the number of pleural eosinophils. In conclusion, our data suggest an important role for resident macrophages and T lymphocytes in the eosinophil accumulation induced by LPS. J. Leukoc. Biol. 56: 151–158; 1994.

Modulation of T lymphocyte and eosinophil functions in vitro by natural tetranortriterpenoids isolated from Carapa guianensis Aublet.

We have previously described the anti-allergic activities of a pooled fraction of tetranortriterpenoids (TNTPs) containing 6α-acetoxygedunin, 7-deacetoxy-7-oxogedunin, andirobin and methyl angolensate isolated from the seeds of Carapa guianensis. In the present study, we performed in vitro studies in order to elucidate the mechanisms by which TNTPs present their anti-allergic effects and to identify the bioactive compound(s) present in such fraction. Here, we show that in vitro incubation of eosinophils with the pooled TNTP fraction, as well as with each one of the five isolated tetranortriterpenoids, impaired the adhesion of eosinophils to tumor necrosis factor-α (TNF-α)-primed tEND.1 endothelial cells. Furthermore, the individual or pooled TNTPs impaired CCL11/eotaxin-mediated chemotaxis. By contrast, pooled TNTPs failed to inhibit adhesion and chemotaxis of T lymphocytes. However, TNTPs were able to impair anti-CD3 monoclonal antibody-induced T cell proliferation and the expression of CD25 and CD69. These data suggest that TNTPs prevent T cell activation. Pretreatment of splenocytes with the pooled TNTP fraction, as well as with each one of the five isolated TNTPs, inhibited ovalbumin (OVA)-induced in vitro production of interleukin-2, chemokine (C-C motif) ligand 11 (CCL11) and regulated on activation normal T cell expressed and secreted (RANTES, also known as CCL5). TNTPs (except 6α-acetoxygedunin) also impaired nuclear factor-κB (NFκB) nuclear translocation in OVA-challenged splenocytes. Taken together, these results demonstrate that the anti-allergic effects of TNTPs isolated from C. guianensis might rely on their ability to inhibit eosinophil migration, as well as the activation of T lymphocytes, which is shared by the five isolated TNTPs.

Synthesis, antimalarial evaluation and molecular modeling studies of hydroxyethylpiperazines, potential aspartyl protease inhibitors, Part 2.

The antimalarial acitivity of hydroxyethylamines, synthesized from the reaction of intermediated hydroxyethypiperazines with benzenesulfonyl chlorides or benzoyl chlorides, has been evaluated in vitro against a W2 Plasmodium falciparum clone. Some of the nineteen tested derivatives showed a significant activity in vitro, thus turning into a promising new class of antimalarials. In addition, a molecular modeling study of the most active derivative (5l) was performed and its most probable binding modes within plasmepsin II enzyme were identified.