Claudio Rugarli

Macrophages exposed to Mycobacterium tuberculosis release chemokines able to recruit selected leucocyte subpopulations: focus on γδ cells

Granuloma is a typical feature of tuberculosis. We evaluated the chemotaxis of selected human leucocyte subsets induced by macrophages incubated with Mycobacterium tuberculosis (MT)‐derived products in vitro. The release of monocyte chemotactic protein 1 (MCP‐1) and interleukin‐8 (IL‐8) correlated with the specific induction of strong chemotaxis towards monocytes and polymorphonuclear leucocytes (PMNs). γδ and T helper type 1 (Th1) αβ lymphocytes were chemoattracted, while T‐resting, IL‐2‐activated and Th2 lymphocytes were unaffected. Activation with mycobacterium‐derived, phosphate‐containing components, modulated the chemokine receptor profile of γδ T lymphocytes as well as their pattern of cyto‐chemokine production, disclosing a potential for their active participation in granuloma formation. In particular, CXCR3 and IP‐10, which we found to be released by MT‐pulsed alveolar macrophages, seem to represent the receptor–counter‐receptor pair implicated in the chemotaxis of γδ lymphocytes. Immunohistochemical analysis and in situ hybridization revealed the in vivo presence of IL‐8, MCP‐1 and IL‐10 in lymph node and lung tuberculous granulomas. Our results underscore the role of MT extracts in the induction of macrophage‐derived chemokines responsible for the orchestrated recruitment of PMNs, monocytes, and Th1 and γδ T cells, as well as in the regulation of γδ function.

Prognostic factors for survival in metastatic renal cell carcinoma: Retrospective analysis from 109 consecutive patients

OBJECTIVE Metastatic renal cell cancer (RCC) portends a bad prognosis, but survival is quite different among different patients. The objective of this study was to determine prognostic factors for survival with the aim to offer patients proper therapeutic options. METHODS A consecutive series of 109 metastatic RCC patients admitted to our department since 1988 was reviewed, and survival from the time of diagnosis with metastases recognition was considered. The role of age, sex, disease-free interval (DFI), ECOG performance status (PS), stage at diagnosis, grading, number and type of metastatic sites, nephrectomy, blood levels of hemoglobin, creatinine, albumin, calcium, lactate dehydrogenase (LDH), ferritin, alkaline phosphatase, triglycerides was assessed in univariate and multivariate analysis. RESULTS In our study, the following variables were found to be statistically significant at the univariate analysis (p < 0.01): DFI, ECOG PS, stage at diagnosis, grading, nephrectomy, sites of metastases, blood hemoglobin, serum albumin, calcium, LDH, alkaline phosphatase. Indeed, only an ECOG PS of 2-3 (relative risk 1.82; p = 0.003) and blood hemoglobin levels < or = 10 g/100 ml (relative risk 1.20; p = 0.017) retained their value as independent risk factors for poor survival at multivariate analysis. According to the number of independent risk factors, three groups of patients were identified, with significantly different median survival (21.7 vs. 8.6 vs. 3.5 months; log-rank test: p = 0.00004, p = 0.04126 and p = 0.00047, respectively). CONCLUSIONS Poor performance status and anemia at diagnosis of metastatic RCC predict the worst outcome in our series. These factors could be taken into account to stratify patients in clinical trails and to select the proper treatment option in oncological practice.

Mycobacterium tuberculosisexploits the CD95/CD95 ligand system of γ δ T cells to cause apoptosis

Vγ9/Vδ2+ T cells specifically recognize Mycobacterium tuberculosis in vitro and are precociously recruited in early mycobacterial lesions. Even if γ δ T cells are only fortuitously detected in granulomas or bronchoalveolar lavages of patients with active pulmonary tuberculosis, a role in shaping the mature α β T cell response against M. tuberculosis is substantiated. Here we provide a molecular explanation for this paradox: the engagement of the γ δ TCR by mycobacterial antigens induced the expression of CD95 ligand (CD95L) by chronically activated CD95+ /CD95L− γ δ T lymphocytes. The receptor was functional, as CD95/CD95L interaction triggered the bystander death of CD95+ cells by apoptosis. Cell death was abolished by CD95‐blocking antibodies. The transient accumulation at the site of infection of CD95L+ γ δ lymphocytes, capable of interacting with CD95+ leukocytes attracted by the response towards the pathogen, may determine the characteristics of the ensuing granulomatous disease.

In vivo administration of GM-CSF promotes the clearance of apoptotic cells: effects on monocytes and polymorphonuclear leukocytes.

The clearance of apoptotic cells is crucial to avoid chronic inflammation and autoimmunity. Little is known about the factors that regulate it in vivo. We show that granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) administration to carcinoma patients confers to their leukocytes a significantly higher ability to phagocytose apoptotic cells than before (P < 0.005). GM‐CSF increased the concentration of monocytes and polymorphonuclear leukocytes in the peripheral blood and activated circulating polymorphonuclear leukocytes. Both effects abated early after treatment, whereas phagocytosis of apoptotic cells was still significantly higher after 18 days compared with basal values (P < 0.005 and P < 0.025 for monocytes and polymorphonuclear leukocytes, respectively). On in vitro phagocytosis of apoptotic cells monocytes, but not polymorphonuclear leukocytes, up‐regulated MHC class II membrane expression. These findings are consistent with the possibility that GM‐CSF endows both scavenger and antigen‐presenting leukocytes with the ability to internalize apoptotic tumor cells. J. Leukoc. Biol. 67: 174–182; 2000.

Blockade of the Fas-triggered intracellular signaling pathway in human melanomas is circumvented by cytotoxic lymphocytes.

Fas and Fas ligand (FasL) have been found both in lymphoid and in non‐lymphoid malignancies, and are thought to play a role in the interplay between tumors and the immune system. Here we investigated Fas/FasL expression, function and intracellular signalling pathways in human melanomas. Of 5 melanoma cell lines, 3 expressed Fas at their surface, and all of them expressed FasL. FasL was functional, since it triggered Fas‐induced apoptosis of human T lymphocytes clones. Conversely, cross‐linking of Fas molecule with a specific monoclonal antibody failed to induce apoptosis in any of the melanomas tested, or ceramide intracellular accumulation or caspase‐3 activation, pointing to an early alteration in the Fas‐triggered signaling cascade. All melanomas retained the ability to undergo apoptosis induced by cytotoxic lymphocytes, which was mediated by the granule exocytosis mechanism. This suggests that melanoma cells evade immune‐mediated Fas‐triggered apoptosis via a selective blockade of the Fas apoptotic pathway. Cytotoxic lymphocytes, however, may circumvent tumor resistance to Fas‐induced death via granzyme‐mediated apoptosis, further supporting the development of immunotherapeutic strategies in the treatment of cancer. Int. J. Cancer 81:573–579, 1999.

Treatment of advanced renal cell cancer with sequential intravenous recombinant interleukin-2 and subcutaneous α-interferon

Starting from in vitro studies suggesting synergistic antitumour activity against renal cell cancer (RCC) of recombinant interleukin-2 (rIL-2) and alpha-interferon (IFN), a phase II trial was initiated to test the clinical activity of this combination. The two cytokines were administered sequentially, with the aim of reducing the risk of additive toxicity and enhancing the immunological reaction against the tumour. The original treatment schedule consisted of rIL-2 18 x 10(6) U/m2/day by continuous intravenous infusion for 120 h days 1-5, and alpha-IFN 2b, at a flat dose of 9 x 10(6) U by subcutaneous or intramuscular injection thrice in a week, from day 8 to 28. Treatment was planned to be continued for six or more 28-day cycles, depending on clinical response. 12 patients were treated according to this schedule; as some cardiovascular toxicity was experienced in this set of patients, 11 further patients were treated with half-dose rIL-2 (i.e. 9 x 10(6) U/m2/day). 17 out of 23 enrolled patients completed at least one cycle of treatment and were evaluated for response. We observed six major responses [one complete response (CR) + five partial responses (PR)] for an objective response rate of 35% [95% confidence interval (CI) 17-59%]. 5 additional patients achieved stabilisation of disease; one of them reached CR after surgical extirpation of a lung mass. Sites of response included lung, nodes and bone. Duration of response is 12+ months for CR; 17, 16, 12+, 9 and 9 months for PRs. Median survival is 16 months. Response was not significantly different between full-dose and half-dose rIL-2. Considering stable disease (SD) as responses, there seemed to be a higher chance of response for patients with smaller tumour burden (P = 0.032). The toxicity of rIL-2 treatment, mainly cardiovascular, was substantial; 9 patients experienced severe cardiotoxicity, consisting of major arrhythmias, myocardial ischaemia, reduction of ejection fraction measured with heart radionuclide scan, and were excluded from continuing treatment. Other rIL-2-related toxicities forcing exclusion from the study were severe thrombocytopenia (1 case), and generalised exfoliative dermatitis requiring steroids (1 case). Otherwise, treatment was well tolerated; rIL-2-related toxicities promptly recovered after rIL-2 discontinuation in the majority of cases, and no treatment-related deaths were reported. The half-dose rIL-2 regimen was significantly less toxic in terms of hypotension (P = 0.014), fever (P = 0.014), oliguria (P = 0.042), serum creatinine elevation (P = 0.009) and prothrombin time elongation (P = 0.038).(ABSTRACT TRUNCATED AT 400 WORDS)

Melanoma cells interfere with the interaction of dendritic cells with NK/LAK cells.

Dendritic cells (DCs) and natural killer (NK) cells are key players at the interface between innate resistance and acquired immunity. NK cells can induce DC maturation, a differentiation process whereby DCs respond to a environmental stimulus and acquire the ability of eliciting adaptive immunity. Conversely, maturing DCs promote NK functions in vivo and in vitro. This interplay has important consequences on the immune response to pathogens and possibly to neoplastic cells. Here, we show that B16 melanoma cells actively modulate the interaction between DCs derived from bone marrow precursors and NK/LAK cells propagated from the spleen of C57BL/6 mice. DCs increased in a dose‐dependent manner the ability of NK/LAK cells to kill melanoma cells and to produce cytokines. This activatory cross‐talk entailed the production of IL‐18 by DCs and of IFN‐γ by NK/LAK cells. Melanoma cells were not a passive target of NK activity; they regulated the outcome of the interaction between DCs and NK/LAK cells, inhibiting the in vitro production of cytokines as effectively as the genetic deletion of IL‐18 or IFN‐γ. Interference with the NK/DC interaction possibly represents a mechanism used by growing tumors to evade the immune response.

Melanoma and lymphoma rejection associated with eosinophil infiltration upon intratumoral injection of dendritic and NK/LAK cells.

Dendritic cells (DCs) are promising tools for tumor immunotherapy. Their efficacy in the tumor environment increases when tumor cells die as a consequence of chemo/radiotherapy or when local stimuli promoting DC maturation and function are available. Dying tumor cells could represent a source of tumor antigens, which DCs cross-present to tumor-specific T cells. The outcome of cross presentation is in turn determined by the maturation state of DCs. Natural killer (NK)/lymphokine-activated killer (LAK) cells injected into growing tumors could both provide a source of dying cells for cross-presentation and deliver stimuli for DC maturation. Here, we report that NK/LAK cells recognized and killed in vivo major histocompatibility complex class Ilow highly tumorigenic, nonimmunogenic B16F1 melanoma cells when injected into exponentially growing neoplastic lesions. The simultaneous injection of immature DCs was required to heal animals. Similar results were obtained injecting NK/LAK cells and DC into growing Raucher leukaemia virus induced cell line lymphomas. Cured mice failed to reject other implantable tumors, and developed a specific cytotoxic response against the original neoplasm; moreover, they developed a long-lasting memory, and were protected against further challenges with living tumor cells only when both cell populations were introduced. The response associated to the preferential recruitment within tumors of eosinophils. The simultaneous injection in solid tumors of DCs and NK/LAK cells represents an attractive approach for antineoplastic immunotherapeutic strategies.

Cytokine secretion associated with the clearance of apoptotic bodies in renal cell carcinoma patients

The factors determining the outcome of immunotherapy in metastatic renal cell carcinoma (RCC) patients remain elusive. Macrophages from normal donors that phagocytose apoptotic cells secrete the immunosuppressive cytokine IL‐10 in vitro. Conversely, IL‐10 genetic deletion enhances the immunogenicity of apoptotic tumor cells in vivo. Elevated pre‐treatment levels of IL‐10 are associated with an unfavorable outcome of RCC. We examined whether the ability to release IL‐10 by macrophages from RCC patients that phagocytosed apoptotic cells correlated with the outcome of immunotherapy. To this aim, we derived macrophages from 30 patients with metastatic RCC and from 21 healthy subjects (11 sex‐ and age‐matched healthy controls and 10 younger donors). Patients either had a clinical response after immunotherapy, with a median survival after treatment of more than 18 months (n = 16), or were beginning immunotherapy after diagnosis of metastatic disease (n = 14). Macrophages from responding patients challenged with apoptotic cells released significantly less IL‐10 than controls (p = 0.0075) and recently diagnosed patients (p = 0.0198), as ascertained by a 2‐sided Students t‐test. This was not because macrophages from responding patients lost the ability to secrete IL‐10, because antibody opsonization of apoptotic cells rescued IL‐10 secretion. In contrast, macrophages from all groups of donors released similar amounts of TNF‐α. The failure in IL‐10 secretion by engulfing macrophages of responding subjects may exalt the immunogenicity of dying tumor cells, contributing to the success of immunotherapy.

Effect of cyclosporine and aminophylline on streptozotocin-induced diabetes in rats

Diabetes induced in rats by multiple low doses of streptozotocin is thought to mimic type 1 disease in man. We tested the effect of concomitant treatment with immunomodulator drugs in this diabetic experimental model. Administration of cyclosporine resulted in a rapid appearance of hyperglycemia, perhaps by a potentiation of the direct cytotoxic action of streptozotocin on beta cells. By contrast, aminophylline administration protected the animals from the diabetogenic action of streptozotocin. Concomitant treatment with aminophylline and cyclosporine failed to protect the rats from the hyperglycemia induced by streptozotocin.