Injection by various routes of melanoma antigen-associated macrophages: biodistribution and clinical effects

Patients' autologous macrophages (AM) were used as antigen-presenting cells (APC) in a vaccination protocol against malignant melanoma. AM were administered by various routes, including intralymphatic, since these cells did not express CCR7, a molecule required for APC migration to lymph nodes. Seven HLA-A2 patients with metastatic melanoma-two classified as M1 and five as M3-were included in the study. AM were produced from leukapheresis-separated mononuclear cells by 7-day culture with granulocyte-macrophage colony-stimulating factor. After separation by elutriation, AM were frozen in aliquots and subsequently thawed at monthly intervals, exposed to MAGE-3(271-279) peptide and injected subcutaneously into lymph nodes or into one peripheral lymph vessel. Intradermal tests were performed before and after treatment to determine peptide reactivity. No acute toxicity was observed following injection. One M1 patient had a 7-mm induration intradermal reaction response and was stabilized for 64 weeks. The M3 patients did not show any immunological or clinical response. In 11 patients, the biodistribution of 111In-labeled AM was investigated. There was no clear evidence that AM injected intradermally or subcutaneously left the site of injection. After injection into a lymph vessel of the foot region, scintigraphs showed five to ten popliteal and inguinocrural lymph nodes. This appeared to be the most efficient way to administer rapidly and safely large amounts of peptide-loaded APC into lymph nodes.     

Conversion of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus (ST:FeSV): tumoricidal potential in monolayers and in agar suspensions.

In earlier studies [1-3], we have demonstrated the conversion of human fibroblasts (HF) to tissue macrophages (TM) by the Snyder-Theilen feline sarcoma virus (ST:(FeSV)). The purpose of the present study is to determine the cytolytic potential of ST:FeSV(FeLV)-induced TM against tumorigenic target cells under defined conditions in vitro. The results show that ST:FeSV-induced TM, but not mock-infected HF, produced significant lysis of human colon adenocarcinoma cells (LS-180) after a 3-day preincubation period, followed by a 4-day coincubation period at an effector to target cell ratio of 5:1. The presence of IFN-gamma, or lipopolysaccharides (LPS), and especially of M-CSF, during the coincubation period generally yielded optimal lysis of the tumor cells. Addition of LS-180 specific antibody (NRCO-4) substantially increased the cytolytic potential of TM. Significantly, coincubation of TM with LS-180 tumor cells in an agar medium, where no direct contact between cells occurs, resulted in the inhibition of tumor cell proliferation. Addition of LPS has further accentuated this inhibition. The results indicate that ST:FeSV-induced macrophages are potent oncocytolytic agents of LS-180 tumor cells in the absence and in the presence of direct contact between effector and target cells.     

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon gamma (IFN-gamma) is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alpha) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alpha, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.     

Angiotensin II-induced mononuclear leukocyte interactions with arteriolar and venular endothelium are mediated by the release of different CC chemokines

Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1alpha generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80-90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 antagonist, or an anti-MCP-1 antiserum, without affecting the increased endothelial expression of P-selectin and VCAM-1. Conversely, leukocyte interactions with the venular endothelium, although inhibited by Met-RANTES, were little affected by the anti-MCP-1. Using rat whole blood in vitro, Ang-II (100 nM) induced the expression of monocyte CD11b that was inhibited by Met-RANTES but not by anti-MCP-1. Stimulation of human endothelial cells (human umbilical arterial endothelial cells and HUVECs) with 1-1000 nM Ang-II, predominantly acting at its AT(1) receptor, induced the release of MCP-1 within 1 h, RANTES within 4 h, and MCP-3 within 24 h. Eotaxin-3, a natural CCR2 antagonist, was released within 1 h and may delay mononuclear cell responses to MCP-1. Therefore, Ang-II-induced mononuclear leukocyte recruitment at arterioles and venules is mediated by the production of different CC chemokines. Thus, Ang-II may be a key molecule in the initial attachment of mononuclear cells to the arterial endothelium in cardiovascular disease states where this event is a characteristic feature.     

An evaluation of the metabolic basis of aflatoxin B1 toxicity by using buffalo granulocytes and agranulocytes in vitro

This study was aimed at monitoring cytotoxic changes in buffalo leukocyte subpopulations exposed to aflatoxin B1 (AFB1), since no such information is available for this species. The effects of AFB1 on glutathione (GSH) S-transferase, Ca2+Mg2+ATPase and protein synthesis in leukocyte subpopulations, namely, mononuclear (MN) cells and polymorphonuclear (PMN) cells isolated from the blood of the domestic buffalo (Bos bubalis), were studied. The cells were separated by using Ficoll-Paque and incubated in the presence of AFB1. GSH S-transferase activity was found to increase in cells exposed to AFB1, but there was no difference in activity between MN and PMN cells. PMN cell ATPase activity increased after AFB1 treatment, whereas no such effect was observed in the MN cells, which showed higher basal levels of ATPase activity. In the presence of AFB1, all the cells showed significant decreases in 14C-leucine incorporation, but the MN cells showed higher 14C-leucine incorporation than the PMN cells. Nevertheless, both cell types were affected by AFB1 and participated in its detoxification. There was also an appreciable decrease in the release of myeloperoxidase by activated PMN cells in the presence of AFB1.     

Inflammatory responses in experimental tuberculosis pleurisy

A model of tuberculous pleurisy in New Zealand white rabbits was developed to describe the sequential cellular and biochemical changes in pleural fluid. Bacille Calmette-Guérin (BCG) in 4 X 10(7) colony-forming units was introduced into the right pleural space of rabbits previously sensitized by intradermal BCG. Pleural fluid was obtained via serial thoracenteses. A normal-pH, normal-glucose, exudative effusion was seen through 144 hours. Polymorphonuclear leukocytes were the first cells to respond to the introduction of tubercle bacilli in the pleural space; they remained the predominant cell for the first 24 hours and were followed by macrophages, which peaked at 96 hours, and then by lymphocytes. Numerous granulomata were observed on both the visceral and parietal pleura ten days following intrapleural instillation of BCG. We propose that the polymorphonuclear leukocyte influx is not a nonspecific response to pleural injury and that such a leukocyte response, either itself or through its interaction with the macrophage, plays a role in host defense mechanisms against the tubercle bacilli.     

Cytokines involved in the immunosuppressor period in experimental fasciolosis in rats

The aim of this study was to evaluate the kinetics of the cytokines interferon-gamma, interleukin-2, interleukin-10 and interleukin-4 produced by spleen mononuclear cells stimulated by Con A during an experimental infection in rats with Fasciola hepatica. The proliferative response to Con A of Spm cells from rats infected with F. hepatica was significantly decreased on day 7 post-infection (P<0.006) and simultaneously an increase of interferon-gamma, interleukin-10 and interleukin-4 production along with a decrease of interleukin-2 by spleen mononuclear cells were observed. Interleukin-4 and interleukin-10 were involved in ablating cellular proliferation in vitro, as the addition of neutralising antibodies to either cytokine reversed the proliferative block. The addition of exogenous recombinant interleukin-2 also restored the proliferative response by spleen mononuclear cells obtained 7 days after infection from infected rats. At the same time, we found an increase in interleukin-10 production by peritoneal cells (in close contact with the flukes) and decreased nitric oxide levels. In addition, histological studies on the liver on day 7 after infection showed the presence of parasite inside migratory tunnels in the parenchyma, and polymorphonuclear leukocytes, predominantly eosinophils, around the parasite. The transient suppression in proliferative response mediated by cytokines interleukin-4 and interleukin-10 in the spleen, and diminution of nitric oxide production in the peritoneum could be mechanisms to evade the protective immune response during the first stages of liver penetration by the parasite.     

Effect of protein kinase C activators on the phosphorylation and the surface expression of the CDw50 leukocyte antigen.

The CDw50 antigen is a constitutively non-phosphorylated leukocyte surface molecule which becomes highly phosphorylated in all the normal and lymphoblastoid cells analyzed (peripheral blood mononuclear cells, Molt 4, CEM, 8402, Namalwa), after stimulation with tumor promoter agents (phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, mezerein). This phosphorylation is rapid (within 1-5 min), dose-dependent and results in the incorporation of PO(3-)4 groups on serine residues. Furthermore, the level of CDw50 phosphorylation induced by tumor promoter agents is decreased by the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. Activation of peripheral lymphocytes with concanavalin A, phytohemagglutinin and cross-linking of CD3 molecules also induces CDw50 phosphorylation, but the response is delayed and less intense than when tumor promoting agents are used. Treatment with any of the aforementioned agents is not accompanied by quantitative changes in the CDw50 surface expression. We therefore conclude that protein-kinase-C-mediated mechanisms are involved in phosphorylation, but not in regulation of the surface expression of the CDw50 leukocyte antigen.     

Differential inhibition of polymorphonuclear leukocyte recruitment in vivo by dextran sulphate and fucoidan

The selectin-mediated rolling of leukocytes along the endothelial cells is a prerequisite step followed by firm adhesion and extravasation into the inflamed tissue. This initial contact can be suppressed by sulphated polysaccharides. We have studied the effect of sulphated polysaccharides on the ultimate polymorphonuclear leukocyte (PMN) recruitment and plasma leakage in rabbit skin in response to intradermal injection of various inflammatory mediators. PMN infiltration evoked by various PMN chemoattractants (FMLP, C5a desArg, LTB(4) and IL-8) was significantly inhibited after intravenous injection of dextran sulphate (25 mg/kg), heparin (2 x 90 mg/kg) or fucoidan (1 mg/kg). PMN-dependent plasma leakage was equally well reduced by the different sulphated polymers. Vascular permeability induced by histamine or thrombin acting via a PMN-independent mechanism was not reduced. Fucoidan was the only polysaccharide able to suppress IL-1-induced PMN infiltration for 60-70%. Local administration of dextran sulphate had no effect on PMN-dependent plasma leakage. Differential inhibition of PMN recruitment was determined after injection of dextran sulphate or fucoidan depending on the type of insult. Therefore, these results suggest that different adhesion pathways are utilized during PMN recruitment in vivo in response to chemoattractants and IL-1.     

Arachidonic acid metabolism among human mononuclear leukocytes. Lipoxygenase-related pathways

Human peripheral blood mononuclear cells were isolated and assessed for the presence of contaminating polymorphonuclear leukocytes and platelets. Incubations of these cell isolates were performed in the presence or absence of the calcium ionophore A23187 and/or 1-14C-labeled or unlabeled arachidonic acid. Using reverse phase high pressure liquid chromatography with simultaneous monitoring of ultraviolet light absorption at 229 and 280 nm and, where appropriate, of radioactivity, our studies reveal that human peripheral blood mononuclear cells generate leukotrienes C4 and B4 (LTC4 and LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) following stimulation with A23187. The ratio of LTC4 to LTB4 was approximately 10-fold greater among the mononuclear cells than among similar incubations of polymorphonuclear leukocytes. Furthermore, the mononuclear cells failed to metabolize LTB4 into the omega-hydroxy or omega-carboxy derivatives that were always present in, and very characteristic of incubations of polymorphonuclear leukocytes. Depletion of monocytes from the mononuclear cells by double adherence resulted in virtual loss of the generation of 5-lipoxygenase-derived products by the remaining nonadherent cells, supporting the conclusion that the monocytes and not the lymphocytes were the source of LTC4, LTB4, and 5-HETE. The presence of both 12-HETE and the cyclooxygenase-derived 12-hydroxyheptadecatrienoic acid correlated with the degree of platelet contamination, suggesting that the platelets account for the presence of these compounds.     

Molecular mechanisms underlying IL-4-induced leukocyte recruitment in vivo: a critical role for the alpha 4 integrin.

IL-4 is known to induce recruitment of eosinophils and mononuclear leukocytes. In vitro this occurs in part by selective expression of VCAM-1, the ligand for the alpha 4 integrin. The objective of this study was to determine the molecular mechanisms that underlie IL-4-induced leukocyte recruitment in vivo. Mice received an intrascrotal injection of IL-4 (100 ng). Twenty-four hours later, leukocyte rolling, adhesion, and emigration in cremasteric postcapillary venules were examined via intravital microscopy, and expression of VCAM-1 and P- and E-selectin was quantitated using a radiolabeled mAb technique. IL-4 increased VCAM-1 expression, but P-selectin and E-selectin remained at constitutive levels. IL-4 induced significant increases in leukocyte adhesion and emigration, with 50% of the emigrated cells being eosinophils and the remainder being mononuclear leukocytes. Leukocyte rolling in IL-4-treated mice was >95% inhibitable using an anti-P-selectin Ab. However, IL-4-induced leukocyte recruitment was unaltered in mice treated chronically with P-selectin Ab or mice deficient in either P-selectin or P- and E-selectin, suggesting that the residual rolling supported all of the IL-4-induced recruitment. In IL-4-treated mice following P-selectin blockade, tethering and rolling were not dependent on L-selectin, but were abolished by alpha 4 integrin blockade. These findings show that the alpha 4 integrin can initiate leukocyte-endothelial cell interactions in the absence of selectins under shear conditions in vivo, and that the absence of selectins does not affect recruitment of eosinophils and mononuclear cells to IL-4-treated tissue.     

Beta-adrenergic modulation of the polymorphonuclear leukocyte respiratory burst is dependent upon the mechanism of cell activation

Although inhibition of polymorphonuclear leukocyte activation by beta-adrenoceptor agonists has been recognized for over a decade, effects have only been observed at high drug concentrations and in the presence of theophylline. In this study, catecholamine and prostaglandin modulation of the respiratory burst was evaluated with respect to the mechanism of polymorphonuclear leukocyte activation. Very low concentrations of isoproterenol and prostaglandin E2 inhibited the respiratory burst when induced by chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) or calcium ionophore (A23187, ionomycin), but not when initiated by synthetic diacylglycerol. Because formyl-methionyl-leucyl-phenylalanine and ionophore mobilize calcium and arachidonic acid generation follows an increase in intracellular calcium, the arachidonic acid metabolite leukotriene B4 was studied. Isoproterenol at a very low (0.1 nM) concentration also rapidly inhibited leukotriene B4 generation. Since cyclic AMP was increased by isoproterenol regardless of the means of cell activation, modulation of intracellular calcium was evaluated with the fluorescent probe indo-1. A transient increase in calcium after formyl-methionyl-leucyl-phenylalanine or ionophore (but not oleoyl acetylglycerol) cell activation was inhibited by isoproterenol or prostaglandin E2. These results suggest that adrenergic agonists specifically modulate calcium-dependent polymorphonuclear leukocyte function. Because marked inhibition was observed at very low drug concentrations, cyclic AMP-dependent effects may be important in both homeostatic and therapeutic modulation of inflammatory response.     

Suppression of mitogen- and tumor-cell-induced lymphocyte stimulation by tumor-associated fetal antigens

The ability of tumor-associated fetal antigens (TAFA) to suppress mitogen and tumor-cell-induced blastogenesis was investigated. Three different TAFA (I, III, and IV) were tested in PHA and Con A lymphocyte proliferation assays. Spleen cells from New Zealand black rats (NBR) were fractionated over nylon wool; and nonadherent (NA) and adherent (AD) cells were compared with unfractionated (UF) cell responses. Preincubation of NA cells with TAFA-I was followed by addition of phytohemagglutinin (PHA) elicited suppression in a 3-to 4-day assay. UF cells were unresponsive to TAFA and/or PHA at all concentrations tested. TAFA dose—response titration curves in Con A proliferation assays revealed that all TAFA tested (TAFA I, -III, and -IV from fibrosarcomas; TAFA-I and -III from osteosarcomas) were suppressive. For some TAFA, nanogram quantities produced significant suppression. In mixed leukocyte tumor cell assays (MLTC) both UF and NA normal rat spleen cells were tested for proliferative responses to syngeneic tumor cells. Four distinct TAFA, purified by high-pressure liquid chromatography, suppressed lymphocyte proliferation when added to MLTC cultures in 5-day assays. Specificity experiments demonstrated that trinitrophenol-bovine serum albumin did not produce similar immunosuppression. TAFA did not block recognition of tumor antigen nor produce nonspecific cytotoxicity of the spleen cells. Significant suppression of DNA synthesis was produced by TAFA-1 following cocultivation with spleen and tumor cells for 1, 2, and 3 days, compared to no suppression when spleen and tumor cells were washed free of TAFA-I prior to tumor cell addition at Day 0. Similar experiments using rat embryo fibroblasts (REF) as stimulators demonstrated that pre-REF cocultivation treatment of lymphocytes with TAFA-I significantly reduced subsequent blastogenic responses. This effect was not reversible; however, if TAFA-I was added to responders previously stimulated by REF, a suppressive response was not seen. Experiments were also carried out to determine the reversibility of TAFA-I effects. Cells were treated with TAFA-I from 1 to 5 days, followed by determination of lymphocyte blastogenesis. TAFA-I effects are reversible and antigen presence is required to completely suppress (or inhibit) stimulation by tumor cells.     

Macrophage procoagulant-inducing factor. In vivo properties and chemotactic activity for phagocytic cells

Murine macrophage procoagulant-inducing factor (MPIF) is a lymphokine with chemical properties distinct from a number of well-characterized cytokines. MPIF induces procoagulant activity on the surface of macrophages and thus may play a central role in the expression of cell-mediated immunity. Highly enriched MPIF-alpha and -beta, separated by virtue of their basic isoelectric point and affinity for heparin, induced local induration and fibrin deposition and cellular infiltration similar to that observed in delayed type hypersensitivity reactions, when injected intradermally. Margination with of polymorphonuclear leukocytes (PMN) along the endothelium as well as increased PMN infiltration was evident after 4 h. In contrast to other inflammatory mediators (e.g., C5a, IL-1) reactivity was sustained, with greater numbers of mononuclear cells apparent 24 h after skin testing. Changes in the dermis were evident 4 h after MPIF injection with increased numbers of cells near areas where spaces in the collagen bundles had formed. Dermal thickening was evident after 24 h and collagen fiber structure was disrupted. Extravascular fibrinogen/fibrin was most prominent 24 h after testing. LPS, which induces macrophage procoagulant activity in vitro, did not induce the histopathologic changes evident with MPIF. MPIF was chemotactic for PMN and macrophages in vitro. Chemotactic activity was heat-labile and not due to C5a. Migration was dependent on a concentration gradient, as determined by checkerboard analysis, indicating that MPIF promoted chemotaxis rather than chemokinesis. Experiments reported here suggest that MPIF is an important mediator of fibrin deposition and cellular infiltration characteristic of cell-mediated immune response.     

The cutaneous response in humans to Treponema pallidum lipoprotein analogues involves cellular elements of both innate and adaptive immunity

To extend prior studies implicating treponemal lipoproteins as major proinflammatory agonists of syphilitic infection, we examined the responses induced by intradermal injection of human subjects with synthetic lipoprotein analogues (lipopeptides) corresponding to the N termini of the 17- and 47-kDa lipoproteins of Treponema pallidum. Responses were assessed visually and by flow cytometric analysis of dermal leukocyte populations within fluids aspirated from suction blisters raised over the injection sites. Lipopeptides elicited dose-dependent increases in erythema/induration and cellular infiltrates. Compared with peripheral blood, blister fluids were highly enriched for monocytes/macrophages, cutaneous lymphocyte Ag-positive memory T cells, and dendritic cells. PB and blister fluids contained highly similar ratios of CD123(-)/CD11c(+) (DC1) and CD123(+)/CD11c(-) (DC2) dendritic cells. Staining for maturation/differentiation markers (CD83, CD1a) and costimulatory molecules (CD80/CD86) revealed that blister fluid DC1, but not DC2, cells were more developmentally advanced than their peripheral blood counterparts. Of particular relevance to the ability of syphilitic lesions to facilitate the transmission of M-tropic strains of HIV-1 was a marked enhancement of CCR5 positivity among mononuclear cells in the blister fluids. Treponemal lipopeptides have the capacity to induce an inflammatory milieu reminiscent of that found in early syphilis lesions. In contrast with in vitro studies, which have focused upon the ability of these agonists to stimulate isolated innate immune effector cells, in this study we show that in a complex tissue environment these molecules have the capacity to recruit cellular elements representing the adaptive as well as the innate arm of the cellular immune response.     

Relationship of Footpad Sensitivity to Purified Protein Derivatives and Resistance to Airborne Infection with Mycobacterium tuberculosis of Mice Vaccinated with Mycobacterial Cell Walls

Footpads of mice sensitized by oil-treated Bacillus Calmette-Guérin (BCG) cell walls given either intravenously, subcutaneously, intradermally, intraperitoneally, or intramuscularly became swollen and reddened after injection of purified protein derivative (PPD). This reaction, greatest after intradermal and subcutaneous sensitization, generally reached a maximum about 24 hr after challenge and was still marked at 48 hr. The histological response was characterized by infiltration with both polymorphonuclear and mononuclear cells. The proportion of mononuclear cells increased with time and they predominated at 48 hr. The footpad reaction could be detected as early as 1 week after sensitization and persisted for at least 37 weeks. Footpad sensitivity to PPD and acquired resistance to airborne infection with Mycobacterium tuberculosis H37Rv were correlated in that (i) both reached a peak approximately 1 month after sensitization of the mouse, and (ii) cell walls treated with NaOH or given without oil neither protected mice against challenge infection nor sensitized them to PPD. Although, as we reported previously, mice vaccinated subcutaneously or intradermally exhibited little or no enhanced resistance to experimental infection, mice given oil-treated cell walls by these routes were highly sensitive to footpad inoculation of PPD. Therefore, the footpad test cannot be used to determine immunity of the mouse to pulmonary infection with tubercle bacilli.     

Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction.

The effect of glutathione (GSH) depletion by L-buthionine-[S,R]-sulphoximine (BSO) on tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression and mononuclear leukocyte adhesion to human umbilical vein endothelial cells (HUVECs) was investigated. Cells with marked depletion of cytoplasmic GSH, but with an intact pool of mitochondrial GSH, only slightly enhanced TNF-alpha-induced E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression, compared with the control. However, TNF-a-induced expression of both molecules was markedly enhanced when the mitochondrial GSH pool was diminished to <15% of the control. In contrast, TNF-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression was not affected by the depletion of either cytoplasmic or mitochondrial GSH. Marked enhancement of TNF-alpha-induced adhesion molecule expression by the depletion of mitochondrial GSH resulted in increased in mononuclear leukocyte adhesion to treated HUVECs, compared with the control. These effects parallel reactive oxygen species (ROS) formation by the depletion of mitochondrial but not cytoplasmic GSH. Our findings demonstrate that depletion of mitochondrial GSH renders more ROS generation in HUVECs, and mitochondrial GSH modulates TNF-alpha-induced adhesion molecule expression and mononuclear leukocyte adhesion in HUVECs.     

The tumorigenicity of IL-2 gene-transfected murine M-3D melanoma cells is determined by the magnitude and quality of the host defense reaction: NK cells play a major role.

Transfection of a variety of tumor lines with the IL-2 gene strongly reduces their tumorigenic potential when applied to either euthymic or athymic animals. To elucidate the mechanisms underlying this phenomenon, we inoculated IL-2-transfected M-3D melanoma (M-3D-IL-2) cells into DBA/2 mice immunosuppressed by gamma-irradiation. Animals thus treated developed pigmented tumors, suggesting that IL-2 transfection of melanoma cells, instead of altering their neoplastic growth properties, renders them capable of evoking a tumoricidal host response. To define the critical effector cell, we injected M-3D-IL-2 and, for control purposes, nontransfected M-3D cells into DBA/2 recipients and analyzed the injection site. We found that 1) IL-2-expressing M-3D cells induce a much stronger inflammatory reaction than wild-type cells, 2) in both instances the infiltrate consists mainly of macrophages (40-60%) and granulocytes (30-40%), and 3) only the infiltrate of M-3D-IL-2 cell deposits contains a minor fraction of NK cells (approximately 1-2%). When we reconstituted sublethally irradiated animals with various leukocyte subsets, we found that unfractionated as well as macrophage-depleted peritoneal lavage cells but not NK cell-depleted peritoneal lavage cells were able to suppress the growth of IL-2-expressing M-3D cells. In vivo leukocyte depletion experiments showed that the NK cell-depleting asialo-GM1 antiserum, but not anti-macrophage and/or anti-granulocyte reagents, restored the tumorigenicity of M-3D-IL-2 cells. Our results indicate that the inflammatory tissue response evoked by IL-2-transfected cancer cells includes the attraction and/or activation of NK cells and that, in the experimental system used, these cells are critically needed for successfully controlling cancer growth in vivo.