Smac127 Has Proapoptotic and Anti-Inflammatory Effects on Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia. Fibroblast-like synoviocytes (FLSs) are apoptosis-resistant and contribute to the pathogenesis of RA by producing cytokines and proteolytic enzymes, which degrade the extracellular matrix. We evaluated the proapoptotic and anti-inflammatory activity of the small molecule Smac127 on RA-FLSs cultured in synovial fluid (SF), in order to reproduce the physiopathological environmental characteristic of RA joints. In this context, Smac127 induces apoptosis by inhibiting apoptosis proteins (IAPs). This inhibition activates caspase 3 and restores the apoptotic pathway. In addition, Smac127 induces a significant inhibition of the secretion of IL-15 and IL-6, stimulation of pannus formation, and damage of bone and cartilage in RA. Also the secretion of the anti-inflammatory cytokine IL-10 is dramatically increased in the presence of Smac127. The cartilage destruction in RA patients is partly mediated by metalloproteinases; here we show that the MMP-1 production by fibroblasts cultured in SF is significantly antagonized by Smac127. Conversely, this molecule has no significant effects on RANKL and OPG production. Our observations demonstrate that Smac127 has beneficial regulatory effects on inflammatory state of RA-FLSs and suggest a potential use of Smac127 for the control of inflammation and disease progression in RA.

Inhibitory effect of pasireotide and octreotide on lymphocyte activation.

Somatostatin (SST) regulates the function of the central and peripheral nervous system, the endocrine and exocrine organs, as well as the vascular and immune system. These actions are mediated by five specific membrane somatostatin receptors. This study compares the effects on human lymphocytes of two long-acting somatostatin analogues that have different receptor affinity: octreotide and pasireotide. Both analogues have an antiproliferative effect on human lymphocyte proliferation, but they act at different concentration and, while octreotide enhances IL10 and inhibits gamma IFN pasireotide inhibits IL2 and gamma IFN. In both sets of experiment the different behaviour of the two analogues could be due to their different affinity to the SSTR subtypes. Finally this study suggest that the growth inhibitory action of somatostatin analogues is an apoptotic phenomenon and it can be mediated by SSTR2a, in the case of octreotide, and by SSTR3 when pasireotide is used or it can be mediated by the heterodimerization of the two receptor.

Effects of chimeric somatostatin-dopamine molecules on human peripheral blood lymphocytes activation.

BIM 23A761, selective for somatostatin receptors subtypes 2, 5 and the dopamine receptor subtype 2, and BIM 23A757 with affinity for SSTR2 and DAR2 were studied on human PBL proliferation and activation. BIM 23A761 was significantly more potent than specific SSTR and DAR2 agonists in suppressing lymphocyte proliferation induced by mitogen or alloantigen, while BIM 23A757 was more potent than specific SSTR2 and DAR2 agonists in suppressing antigen induced proliferation only. Both molecules displayed enhanced potency in suppressing IFNgamma and IL-6 secretion compared with the SSTR and DAR2 analogs, while only BIM 23A761 was able to inhibit IL-2 secretion and its effect is more potent than the control analogs. Furthermore BIM 23A761 inhibit cell progression into the S phase and then into the G2/M, while BIM 23A757 inhibited bromodeoxyuridine incorporation only during the S phase. Both chimeric molecules resulted significantly more effective than the respective controls.

Serological demonstration of an allogeneic Ia.7 antigen on the cell surface of SJL/J-derived reticulum cell sarcomas.

The reticulum cell sarcomas (RCS) of SJL/J mice are of particular interest since they readily induce the proliferation of syngeneic T-lymphocytes. Previous cellular studies examined the antigens on the RCS which stimulated this response and suggested that the tumor expressed allogeneic I-region-associated (Ia) antigens normally associated with the E alpha:E beta molecular complex (S. M. Wilbur and B. J. Bonavida, Exp. Med., 153: 501-513, 1981). These particular Ia glycoproteins are not expressed on normal SJL/J cells due to a defect in the E alpha polypeptide synthetic pathway. However, the E beta subunit is synthesized normally by these animals but remains intracellular. The SJL/J-derived RCS may circumvent this defect in E alpha subunit biosynthesis. The aberrant synthesis of this polypeptide is thought to allow membrane presentation of an intact pseudoallogeneic Ia glycoprotein which utilized the normally dormant E beta s polypeptide. In the present study, two monoclonal antibodies directed against the Ia.7 specificity of the E alpha chain (13/18, 14-4-4S) were used to examine more directly the expression of this polypeptide on the tumor. Surprisingly, neither antibody was effective against the RCS in a direct complement-mediated cytolysis assay. Nevertheless, the tumor was found to specifically adsorb lytic activity of both the monoclonal antibodies. In addition, both a cold-cell competition assay and indirect immunofluorescence corroborated the data and indicated that the RCS does express detectable levels of the Ia.7 antigen. Normal spleen cells and lipopolysaccharide B-derived blasts from SJL/J mice were found in all experiments to be devoid of any specific reactivity with these monoclonal antibodies. In addition, continued in vivo passage of transplantable RCS was found to cause down-modulation of the Ia.7 specificities on these tumors. Newer RCS transplantable lines, however, expressed demonstrable levels of this alloantigen in both cellular and serological assays. The observed down-modulation could explain the difficulties encountered in defining this specificity on long-term transplantable RCS. In conclusion, the present serological study corroborates the early cell-binding data. An Ia.7 antigen is shown to be expressed on the RCS, yet this specificity could not be detected on normal SJL/J cells.

Chemotherapy and immunotherapy of L1210 leukemic mice with antigenic tumor sublines.

Tumor cells, treated in vivo with anticancer compounds, may acquire new antigenic specificities in addition to any original antigens associated with parental tumors. Treatment of mice carrying the parental leukemias L1210 Ha or L1210 Cr with leukemia cells antigenically altered by treatment with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (L1210 Ha/DTIC and L1210 Cr/DTIC, respectively) was essentially ineffective in prolonging the life span of the animals. However, synergic therapeutic activity was exhibited by administration of L1210 Ha/DTIC cells plus 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of the moderately immunogenic L1210 Ha leukemia and by the combination of L1210 Cr/DTIC cells and lymphocytes immune to L1210 Cr/DTIC administered with 1,3-lymphocytes immune to L1210 Cr/DTIC administered with 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of the low immunogenic L1210 Cr leukemia. Early and advanced L1210 Cr-bearing mice showed marked increases in survival time and a significant number of tumor-free survivors on treatment with cyclophosphamide followed by transfer of lymphocytes immune to L1210 Cr/DTIC cells. When parental tumor cells were used as the immunogen, the therapeutic effect was diminished. Thus, in the current investigation, although immunotherapy per se was essentially ineffective, the immunochemotherapeutic modalities used were successful in markedly increasing the survival time of leukemic animals and resulted in an incidence of cures.

Hematoporphyrin derivative rescue from toxicity caused by chemotherapy or radiation in a murine leukemia model (L1210).

Hematoporphyrin [1,3,5,8-tetramethyl-2,4-bis(hydroxyethyl)-porphin-6,7-dipropio nic acid dihydrochloride derivative] (HPD) is a compound that was studied in a number of laboratories because of its cytocidal activity after activation by light. Modification of immune function seen during the photochemotherapeutic studies prompted attempts to determine the effect of HPD on the immune and hemopoietic systems. Splenic hyperplasia as well as marrow hypercellularity were noted in mice treated with HPD. In vitro phytohemagglutinin or lipopolysaccharide stimulation of spleen lymphocytes caused normal or scant increases in blast transformation compared to the stimulation index for lymphocytes from untreated animals. HPD treatment did not significantly alter production of antibody to sheep red blood cells, as evaluated by hemagglutination or hemolytic assay. In contrast, HPD treatment did promote an increased number of spleen colonies in lethally irradiated mice transfused with syngeneic bone marrow. The capacity of HPD to increase the number of bone marrow and spleen cells has been exploited to accelerate the recovery from peripheral leukopenia induced in animals by previous drug or radiation treatment. The time for full return from severe leukopenia induced by an antimetabolite compound (5-fluorouracil) or an alkylating agent (cyclophosphamide or X-rays was significantly shorter in mice treated with HPD than in controls. Furthermore, improved survival was demonstrated in irradiated mice after HPD treatment. Finally, HPD treatment of L1210 leukemic mice did not affect the antitumor activity of cyclophosphamide. If the properties described here be confirmed, HPD might contribute to recovery of leukopenic cancer patients.

Proapoptotic activity of a monomeric smac mimetic on human fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Inhibitors of apoptosis proteins (IAPs) block cell death in response to diverse stimuli. The mitochondrial protein, second mitochondria-derived activator of caspase (Smac), negatively regulates IAP inhibition of caspase activity. We investigated the proapoptotic activity of a synthetic Smac (Smac 066) on fibroblast-like synoviocytes (FLS) derived from patients with active rheumatoid arthritis (RA). We found that Smac 066 induced significant apoptosis in all RA-FLS samples. Furthermore, IAPs, which are upregulated in RA-FLS, were downregulated by Smac 066. This suggested that IAPs upregulation was responsible for RA-FLS sensitivity to Smac 066. Next, we analysed caspase activation and found that Smac 066 was associated with caspase 8 and caspase 3 activities. We then investigated the mechanism underlying Smac 066 downregulation of IAPs in RA-FLS with an apoptotic pathway array. Interestingly, Smac 066 significantly upregulated IGFBP-5, a protein involved in differentiation, apoptosis, and osteoblastic activation. Smac 066 may represent a new therapeutic approach to RA treatment.

The apoptotic effect of somatostatin analogue SMS 201-995 on human lymphocytes.

The antiproliferative effect of a synthetic octapeptide, somatostatin analogue SMS 201-995 (SMS), and its capacity to bind were evaluated on human peripheral blood lymphocytes (PBL) activated by phytohemoagglutinin (PHA). We then addressed our work to investigate if SMS inhibits PHA activation of PBL by a cytostatic rather than a cytotoxic mechanism. Consequently, we studied the cell cycle distribution and the activation of caspase-3, measuring the presence of the cleavage product of poly(ADP-ribose) polymerases (PARP), and we evaluated the presence of apoptotic DNA by using a monoclonal antibody specific for the single-stranded regions of DNA. All our results indicate that SMS induces apoptosis in activated lymphocytes.

Comparative study of the therapeutic effect of photoactivated hematoporphyrin derivative and aluminum disulfonated phthalocyanines on tumor bearing mice.

Although the hematoporphyrin derivative (Hpd) is one of the most studied photosensitisers for photodynamic therapy (PDT), it is far from ideal. Therefore, many laboratories have been investigating a new group of sensitisers, the phthalocyanines. Particularly, in our laboratory we decided to study the aluminum disulfonated phthalocyanines (AlS2PC). They are chemically stable, readily soluble in water and have a strong absorption in the red part of the spectrum at 675 nm. Mice bearing the MS-2 fibrosarcoma treated with 5 mg/kg of AlS2PC survived indefinitely also using a low laser power of 100 mW/cm2 X 10' of exposure time, in contrast to experiments carried out with Hpd where the optical therapeutic laser power was 400 mW/cm2 X 10' and the dose of Hpd was 25 mg/kg. Furthermore, treatment of mice bearing the highly metastatic tumor, B16 melanoma, with 5 mg/kg of AlS2PC and laser light (100 mW/cm2 X 10'), significantly prolonged the survival time in respect to mice treated with 25 mg/kg of Hpd and laser light (400 mW/cm2 X 10').

Hematoporphyrin derivative photoradiation therapy in murine solid tumors.

The cytocidal activity of light-activated hematoporphyrin derivative (Hpd) in experimental and human tumors is under investigation in many laboratories. This activity is based upon preferential incorporation of Hpd in malignant tissues and its photosensibilization by red light. Treatment of mice bearing MS-2 fibrosarcoma and B16 melanoma, a metastastic tumor, with Hpd and laser light, externally or delivered through a quartz fiber optic imbedded directly into the tumor, significantly prolonged the median survival time. This therapy was compared with surgical excision of primary tumors, and preliminary results on metastatic neoplasm suggest that the photoradiation therapy is more effective than surgery.

Toxicity of fenclor 42 in mice: effects on immunocompetent cells.

The aim of this study is to evaluate the effects of Fenclor 42 (a mixture of trichlorobiphenyls) on the immune system. A prolonged administration of this compound to CD2F1 mice resulted in a reduction of relative spleen and thymus weight according to the dose. Furthermore, spleen weights, total number of splenocytes and relative spleen weights decreased significantly also following a single treatment with 0.5 g/kg or 1 g/kg of Fenclor 42. An analysis of the functional activity of splenocytes pointed out that proliferative response to mitogens was also inhibited. Splenic parameters returned to normal values within 5 days after a single treatment and between 8 and 15 days after a subchronic administration. The functional activity of splenocytes was restored between day +5 and day +8 according to the different schedules of treatment. On the contrary, natural killer cell (NK) activity was never affected by Fenclor 42. Studies are in progress to elucidate the intimate mechanism of the toxicity of Fenclor 42 on immunocompetent cells.

Photodynamic therapy in vitro and in vivo with hematoporphyrin derivative and laser light.

Photodynamic therapy is currently under investigation as a new form of treatment for solid malignant tumors in animals and in humans. The method involves photosensitization of drugs and fluorescent dyes, such as hematoporphyrin derivative (Hpd), after preferential incorporation by neoplastic cells. In in vitro experiments laser light activation completely destroys Hpd-pretreated EL4 cells. Mice bearing MS-2 fibrosarcoma treated with Hpd and laser light survived indefinitely, in comparison with control animals that were untreated or treated only with Hpd or laser light. In mice bearing the highly metastatic tumors B16 melanoma and Lewis lung carcinoma (LLC) treated with Hpd and laser light delivered through a quartz fiber optic significantly prolonged the median survival time. This therapy was compared with surgical excision of primary tumors and, for superficial nonmetastatic neoplasma MS-2, the photodynamic therapy was more effective than surgery, while for metastatic tumors B16 and LLC, there was no significant difference between the two methodologies. However, phototherapy is much less traumatic to the animals.

Dacarbazine-induced immunogenicity of a murine leukemia is attenuated in cells transfected with mutated K-ras gene.

Strong immunogenicity is induced by antitumor triazene compounds in tumor cells through a mutagenic mechanism. A highly immunogenic <> clone, isolated from a dacarbazine-treated L5178Y leukemia of DBA/2 mice, was transfected with K-ras mutated at codon 12 (i.e. ras(m12)). This transfected clone presents at least 2 mutations, one concerning K-ras gene, and the other affecting an unrelated gene, responsible for the generation of a highly immunogenic, MHC class I restricted non-self peptide. The results indicate that cells of <> clone transfected with ras(m12) were less immunogenic than cells of the same origin transfected with the vector alone. Moreover, ras(m12)-transfected cells showed lower levels of H-2K(d) gene expression with respect to those detectable in control cells. In addition, in vivo and in vitro sensitization against <> clone carrying mutated ras did not result in a strong cytotoxic T lymphocyte response against ras(m12)-transfected non immunogenic L5178y target cells. These preliminary results suggest that K-ras mutation could down-regulate the level of tumor immunogenicity, possibly acquired through a mutagenic process affecting other unrelated genes.

Macrophage antitumor activity induced by the antigenic lymphoma L5178Y/DTIC subline.

Murine leukemic cells, after in vivo treatment with antineoplastic drugs, have been shown to express new antigenic specificities that were not detectable on parental cells and that were heritable after the withdrawal of drug treatment. A study was conducted of macrophage antitumor activity triggered by LY/DTIC cells, a subline of LY murine lymphoma, antigenically altered by the drug DTIC. In vitro non-specific inhibition of tumor cell growth was exhibited by spleen and peritoneal macrophages from mice previously challenged with viable LY/DTIC. Peritoneal macrophages from LY/DTIC immune animals showed moderate, although significant lytic activity against unrelated tumor target cells. Supernatants from mixed lymphocyte-tumor cell cultures, in which LY/DTIC immune lymphocytes and LY/DTIC tumor cells had been cultured, rendered normal macrophages non-specifically growth inhibitory for tumor cells.

Induction of new antigenic properties on DTIC-treated L1210 clones.

In vivo treatment of mouse leukemia L1210 with DTIC can induce new antigens on tumor cells that are not detectable on parental cells and that are transmissible as a genetic character. Moreover, L1210/DTIC is rejected by syngeneic hosts. The aim of this study was to investigate whether DTIC selects pre-existing immunogenic clones rather than inducing ex novo new antigenic determinants and to verify the number of induced antigens. L1210 leukemia was cloned in vitro and 4 clones were treated in vivo with DTIC. All the treated clones displayed antigenic properties since they were rejected by syngeneic hosts. Cytotoxic T lymphocytes (CTL) activated against one DTIC clone could recognize and lyse the relevant target. One of these DTIC-modified clones (L4/DTIC) was recloned and the subclones were tested in vivo and in vitro. Two out of six subclones were rejected by syngeneic hosts. CTL specific against these two clones were able to recognize and lyse all the other clones to different degrees. The degree of susceptibility to lysis did not correlate with the capability to evoke an immune response in vivo. Based on these findings we conclude that DTIC does not select pre-existing clones but modifies the tumor cells antigenically, and that the antigenicity induced by DTIC in a cloned tumor line is due to the presence of common antigens shared to different degrees with treated cells.

Down regulation of Qa gene expression on drug-modified tumor cells.

BACKGROUND: Mouse leukemia, L1210, strongly enhances its immunogenicity following in vivo treatment with 5-(3-3'-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC). Previous experiments have shown that transformed cells elicit a cell-mediated response accountable for rejection and resistance to a subsequent injection of parental tumor into a syngeneic host. L1210 expresses classical H-2 class I molecules, and since it has been shown that DTIC treatment does not modify the expression of these molecules, this is a suitable model to study nonclassical class I antigens, such as Qa2 glycoproteins, and their potential role in tumorigenicity. METHODS: Cloned cells from L1210 were treated with DTIC and then H-2D, and Qa antigen expression was studied on four clones, before and after xenogenization with DTIC. RESULTS AND CONCLUSIONS: a strong decrease of Qa2 molecule expression was demonstrated by radioimmunoassay and immunofluorescent staining and was confirmed by FACS and 2D-gel analysis. The presence or the absence of Qa antigens on tumor cells could thus be involved in tolerance or rejection of tumor cells in syngeneic animals.

Monoclonal antibody against human growth hormone receptor.

GHR shows a high degree of homology with the prolactin receptor and with the other receptors that belong to the hemopoietic receptor superfamily. This paper describes a monoclonal antibody (MAb) (2B4B6) specific for both the extracellular domain of human GHR and human growth hormone (GH) binding protein. Mice were immunized against a seven-aminoacid peptide sequence screened by FASTA (sequence similarity search served by Genome-Net) from the European Bioinformatics Institute to exclude the existence of human membrane proteins with significant sequence homology. MAbs were screened against the peptide sequence and 2B4B6 was selected for its capability to recognize the full-length hGHBP. As evaluated by both enzyme-linked immunoadsorbent assay (ELISA) and FACS analysis, this MAb seems to recognize and bind to a hGHR positive cell line, IM-9, as well as a murine cell line, BaF3 (8/6), transfected with a chimeric construct, hGHR/hG-CSFR and expressing hGHR on the cell membrane. Studies investigating the biological effects of this MAb showed that anti-hGHR mediated inhibition of cell proliferation was not due to competition with GH binding but rather to prevention of receptor dimerization. Because of its specificity, this MAb may be usefully applied in situations in which GHR and receptors with a high degree of homology, such as PRLR (prolactin receptor), are expressed simultaneously, as occurs in the immune system.

Monoclonal antibodies against recombinant human growth hormone as probes to study immune function.

Monoclonal antibodies were raised against human recombinant growth hormone (rhGH) and those that did not cross-react with other human recombinant proteins like prolactin (PRL), interleukin 2 (IL-2), insulin, or bovine pituitary growth hormone were selected. The selected hybridoma supernatants were studied for their ability to influence T lymphocyte proliferation when induced either by a mitogen, such as phytohemagglutinin (PHA), or by alloantigen. All supernatants inhibited proliferation. Three MAbs were then purified by several passages on antimouse IgG (or IgM)-agarose columns, and characterized. These MAbs recognized three different epitopes, as revealed by competition study, although their inhibitory effect on PHA-induced T cell proliferation was quite similar. The data demonstrate that the MAbs were not cytolytic, that they did not interfere with the PHA binding to T cell membranes, and, as revealed by FACS analysis, did not bind to the membrane. Finally, these MAbs immunoprecipitated a 44-kDa molecule from PHA-activated T cell-concentrated supernatants. These data indicate that the MAbs recognized a soluble factor that plays a central role in T cell proliferation and that is probably the immune growth hormone.