Feline hybridoma growth factor/interleukin-6 activity

An assay system was developed to measure feline hybridoma growth factor (HGF)/interleukin-6 (IL-6) activity in biological samples containing many kinds of cytokines by using the proliferation of the newly established mouse-rat hybridoma clone, B3B1. The proliferative response of this B3B1 clone was IL-6-specific, and could not be promoted by other cytokines including IL-1, IL-2, IL-3, and granulocyte-colony-stimulating factor (G-CSF). The anti-human B-cell stimulatory factor 2 (BSF-2)/IL-6 antiserum did not neutralize feline HGF/IL-6 activity in conditioned media prepared from feline con A-stimulated splenocytes and unstimulated alveolar macrophages, indicating antigenic differences between species. Feline HGF/IL-6 was eluted into the fractions corresponding to a molecular weight of 30,000-40,000 in gel filtration, and into the fractions at a salt concentration of 0.2-0.3 M NaCl in anion exchange chromatography. The physicochemical properties of feline HGF/IL-6 were slightly different from those of murine and human IL-6.     

IL-6: from laboratory to bedside

In the late 1960s, the essential role of T-cells in antibody production was reported. This suggested to us that certain molecules should be released from T-cells for the stimulation of B-cells. We discovered activities in the culture supernatant of T-cells that induced proliferation and differentiation of B-cells. The factor that induced B-cells to produce immunoglobulins was named B-cell stimulatory factor (BSF)-2. The complementary DNA that encoded human BSF- 2 was cloned in 1986. Simultaneously, interferon-beta2 and 26-kDa protein in the fibroblasts were independently cloned by different groups and were found to be identical to BSF-2. Later, a hybridoma/plasmacytoma growth factor and a hepatocyte-stimulating factor also were proven to be the same molecule as BSF-2. Various names were used for this molecule because of its multiple biological activities, and thereafter, these names were unified as interleukin (IL)-6. Since the discovery of IL-6, rapid progress has been made in understanding its actions, the IL-6 receptor system, and the IL-6 signal transduction mechanism. More importantly, it was involved in numerous diseases, such as rheumatoid arthritis and Castleman's disease. By accumulating the basic knowledge, a new therapeutic approach by blocking IL-6 actions appeared to be feasible for chronic inflammatory diseases.     

IL-6

In the late 1960s, the essential role of T-cells in antibody production was reported. This suggested to us that certain molecules should be released from T-cells for the stimulation of B-cells. We discovered activities in the culture supernatant of T-cells that induced proliferation and differentiation of B-cells. The factor that induced B-cells to produce immunoglobulins was named B-cell stimulatory factor (BSF)-2. The complementary DNA that encoded human BSF-2 was cloned in 1986. Simultaneously, interferon-β2 and 26-kDa protein in the fibroblasts were independently cloned by different groups and were found to be identical to BSF-2. Later, a hybridoma/plasmacytoma growth factor and a hepatocyte-stimulating factor also were proven to be the same molecule as BSF-2. Various names were used for this molecule because of its multiple biological activities, and thereafter, these names were unified as interleukin (IL)-6. Since the discovery of IL-6, rapid progress has been made in understanding its actions, the IL-6 receptor system, and the IL-6 signal transduction mechanism. More importantly, it was involved in numerous diseases, such as rheumatoid arthritis and Castleman’s disease. By accumulating the basic knowledge, a new therapeutic approach by blocking IL-6 actions appeared to be feasible for chronic inflammatory diseases.     

Interleukin 6 (IL-6) in serum and urine of renal transplant recipients.

Hybridoma growth factor (HGF) is a 20-25 kD protein, supporting the growth of hybridoma cells in vitro and capable of replacing feeder cells. It was shown to be produced by human monocytes and a number of cultured cell lines. Recently, HGF was found to be identical to interferon-beta 2 or 26 kD protein and BSF-2, and was renamed interleukin 6 (IL-6). Using a sensitive bio-assay we were able to measure IL-6 activity in the serum and urine of healthy volunteers and renal transplant recipients. Low levels of IL-6 were present in the serum but not in the urine of healthy individuals. In contrast, both serum and urine of renal transplant recipients contained high levels of IL-6 directly after transplantation and during acute rejection episodes. On the basis of kinetic studies of the IL-6 response, it is concluded that serial measurement of IL-6, especially in urine, may be of value in monitoring renal transplant recipients. Moreover, the sensitivity of the bioassay will allow for detailed studies as to the biological significance of IL-6 in health and disease.     

Activation and augmentation of guinea pig macrophages with streptococcal preparation OK-432 and stimulated spleen cell products

A mechanism of macrophage activation by a streptococcal preparation (OK-432) was studied. Peritoneal exudate macrophages from normal guinea pigs treated in vitro with OK-432 were activated, manifesting increased glucose consumption, increased spreading, and morphological alterations under scanning electron microscopy. Macrophages showed extensive spreading within 1 hr after OK-432 treatment; they then became rounded with characteristic ruffles in their surfaces after 6 hr of treatment. Highly purified macrophages were activated as effectively as a crude macrophage preparation, suggesting that the macrophage activation resulted from a direct interaction between macrophages and OK-432. However, when spleen cells were treated with OK-432, a factor(s) capable of activating macrophages was produced in the culture supernatant. Spleen macrophage-rich preparation was found to release the factor(s) upon stimulation with OK-432, but this did not occur with the lymphocyte-or granulocyte-rich preparations. These results indicate that OK-432 not only activates macrophages by direct interaction without lymphokine participation, but also augments the activation by affecting spleen cells, probably macrophages, in such a way as to produce monokines.     

Production of hybridoma growth factor by human monocytes

Human mononuclear leukocytes produce a growth factor (HGF) for hybridoma and plasmacytoma cells. HGF has recently been proven to be identical to IFN-beta 2, 26-kDa protein and BSF-2. HGF can be quantitated in a proliferation assay with the HGF-dependent hybridoma cell line B13.29. By selection of an extremely sensitive variant of this cell line, we were able to measure HGF production of single cells. Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes. There was no need for the monocytes to be stimulated but the cells did require the presence of serum. This serum requirement could be met by purified bovine serum albumin, but not by other proteins like ovalbumin or human gamma-globulin. HGF production in vitro by monocytes starts after 2 h of incubation and is completed within 24 h.     

Streptococcal preparation OK-432-induced interferon in human leukocytes: purification and characterization

Interferon production was induced in leukocyte suspensions from human buffy coats after stimulation with the streptococcal preparation OK-432. At day 2-3 the induced interferon reached a maximal level of 0.9 units/1,000 cells. By a combination of batch adsorption/elution on silicic acid, batch adsorption to DEAE-Sephacel, affinity chromatography on concanavalin A-Sepharose and on poly(U)-Sepharose, this interferon could be purified to a specific activity of 10(7.5) units/mg protein. The antiviral activity was characterized as being solely due to gamma-type interferon by a variety of physicochemical, biochemical and serological criteria. Its molecular weight as determined by gel filtration amounted to 53,000 daltons, and its activity was completely neutralized by highly specific antisera to human gamma-type interferon (45K). The OK-432-induced interferon, as the crude supernatant of stimulated leukocytes, and at several stages of its purification, was found to stimulate the natural killer cell activity of fresh human lymphocytes.     

MAPKs ERK and p38, but not JNK phosphorylation, modulate IL-6 and TNF-α secretion following OK-432 in vitro stimulation of purified human monocytes

Interaction between the immune system and cancer allows for the use of biological response modifiers, e.g. OK-432, in cancer therapy. OK-432, penicillin-killed Streptococcus pyogenes, is used in treating carcinomas, but also lymphangiomas. We have studied the role of monocytes (MOs) in the immune response to OK-432 by examining IL-6 and tumour necrosis factor (TNF)-α secretion after in vitro MO stimulation with OK-432, to some extent in comparison with lipoteichoic acid (LTA) and lipopolysaccharide (LPS). LTA stimulation of whole blood gave IL-6 but not TNF-α secretion, as previously shown with OK-432 stimulation, whereas both cytokines were secreted following LPS stimulation. Addition of the MAPK kinase (MAPKK) MEK inhibitor U0126 inhibited IL-6/TNF-α secretion in a dose-dependent manner. Flow cytometry and to some extent Western blot (Wb) analyses showed that MAPK ERK, located downstream of MEK1/2, is predominantly phosphorylated at isolation from peripheral blood. Addition of the p38 MAP kinase inhibitor SB202190 decreased MO IL-6/TNF-α production upon OK-432 stimulation in a dose-dependent manner. Addition of the MAPK JNK inhibitor SP600125 did not systematically change the MO IL-6/TNF-α OK-432 response. Flow cytometry showed that when stimulating the MOs before isolation from blood, LPS yielded ERK phosphorylation and LPS/LTA p38 phosphorylation, whereas OK-432 had no effects on phosphorylation levels. In conclusion, we have shown that OK-432 resembles TLR2 more than TLR4 stimulation of MOs and depends on MAPKK MEK and MAPK p38, but not on JNK phosphorylation. The MEK and p38 MO OK-432 stimulation dependence is possibly related to the differentiation of cells of the MO lineage.     

Inhibition of autoimmune diabetes in NOD mice with serum from streptococcal preparation (OK-432)-injected mice.

We have recently reported that systemic and chronic administration of recombinant tumour necrosis factor alpha (TNF-alpha), as well as streptococcal preparation (OK-432), inhibits development of insulin-dependent diabetes mellitus (IDDM) in NOD mice and BB rats, models of IDDM. In this study we examined whether serum containing endogenous TNF induced by OK-432 injection could inhibit IDDM in NOD mice. Treatment twice a week from 4 weeks of age with OK-432-injected mouse serum, which contained endogenous TNF (75U), but not IL-1, IL-2 and interferon-gamma (IFN-gamma) activity, reduced the intensity of insulitis and significantly inhibited the cumulative incidence of diabetes by 28 weeks of age in NOD mice, as compared with the incidence in non-treated mice (P less than 0.01) and in mice treated with control serum (P less than 0.02). This inhibitory effect of the serum was diminished, although not significantly, by neutralization of serum TNF activity with anti-mouse TNF antibody. In the mice treated with the serum from OK-432-injected mice, Thy-1.2+ or CD8+ spleen cells decreased (P less than 0.01) and surface-Ig+ (S-Ig+) cells increased (P less than 0.05), whereas the proliferative response of spleen cells to concanavalin A (P less than 0.01) and lipopolysaccharide (P less than 0.05) increased. The results indicate that the inhibition by OK-432 treatment of IDDM in NOD mice was partially mediated by serum factors including endogenous TNF.     

Human immunosuppressive factors produced by T cell hybridoma

A Human OKT8-positive T cell hybridoma was established by using concanavalin A (ConA)-activated peripheral blood mononuclear cells (PBMC) fused with CEM-AGR cells. A culture supernatant of the established T cell hybridoma HI4.2D6 inhibited the mitogen induced proliferation of peripheral blood mononuclear cells (PBMC). It inhibited IL-1-, IL-2, or IL-3-dependent proliferation of each factor-dependent mouse cell lines. While the inhibitory activity of IL-1-induced proliferation was stable, that of IL-2- and IL-3-induced proliferation was abrogated within a week at 4 degrees C. Inhibition of IL-2-dependent growth was also observed using human IL-2-dependent cells, but the growth of other factor-independent human hematopoietic cell lines was not affected at all. By gel filtration, the activity inhibiting IL-2- or IL-3-dependent proliferation was found in the fractions with approximate molecular weight of 18,000 and 100,000.     

Augmentation of interleukin 1 and interleukin 2 production by OK-432

Intraperitoneal (i.p.) administration of OK-432 augmented both interleukin 1 (IL-1) and interleukin 2 (IL-2) production to the rechallenge of OK-432 in vitro. Peritoneal exudate cells (PEC) of mice 8 days after i.p. injection with OK-432 (1 KE/mouse) showed maximum IL-1 production to the restimulation with OK-432 in vitro. OK-432-induced IL-1 was consisted of three molecular weight species (two major peaks: 85 K and 15 K daltons and one minor peak: 67 K daltons) on Sephadex G-100 chromatography. Splenocytes of mice 4 days after i.p. injection with OK-432 (1 KE/mouse) demonstrated maximum IL-2 production to the in vitro rechallenge of OK-432, however, in vivo OK-432 administration failed to enhance ConA-induced IL-2 production in vitro. From gel filtration analysis, OK-432 induced IL-2 had an unique molecular weight (approximately 70 K daltons). From these results, OK-432-induced augmentation of cellular immunity against tumor cells might be due to the activation of so-called lymphokine cascade reaction mediated by IL-1 and IL-2.     

TNF-alpha is secreted by monocytes in transit to become macrophages, but not by peripheral blood monocytes, following OK-432 (lyophilized S. pyogenes) stimulation

OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK-432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK-432 stimulation of purified monocytes gave IL-1beta, IL-1RA, IL-6, IL-12p40 and tumour necrosis factor (TNF)-alpha response. OK-432 stimulation of cells within blood did, however, not yield TNF-alpha secretion. When PBMC or monocytes were cultured in low-attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non-specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK-432. This shows that beta(1-3)-integrin receptor function is not necessary for monocyte OK-432-stimulated TNF-alpha secretion. Direct blockage of the beta(2)-integrin (CD18) receptor by anti-CD18 antibody was also unable to prevent the stimulating effects of OK-432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK-432 stimulation, as shown by Western blot. The Fc-receptor was also ruled out as a main receptor of the OK-432 monocyte response. In conclusion, TNF-alpha secretion is only found in monocytes removed from blood. This TNF-alpha secretion is not mediated through the beta(1-3)-integrin receptors. OK-432 may act as a target-seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK-432 is well suited as a cancer treatment drug.     

Pretreatment of human vascular smooth muscle cells with interleukin-1 enhances interleukin-6 production and cell proliferation (action of IL-1 on vascular smooth muscle cells)

Systemic vasculitis is an inflammatory disorder of blood vessels characterized by a perivascular mononuclear cell infiltration around the vessel and fibrinoid necrosis within vessel walls. Interleukin-1 (IL-1) is a multipotent inflammatory mediator and affects several properties of vascular cells. To determine whether IL-1 could contribute to the pathogenesis of vascular diseases, we examined the effect of IL-1 on B cell stimulatory factor-2/interleukin-6 (IL-6) production by cultured human vascular smooth muscle cells (SMC) and the proliferation of these cells. Supernatants of SMC stimulated IgM synthesis of human B cell line. SKW6-CL4 cells. This activity was increased (1.7 to 2.6-fold) when SMC were pretreated with IL-1 or calcium ionophore A23187 for 48 h, and was completely blocked by rabbit anti-human IL-6 antibodies. These IL-6 activities of the SMC supernatants were also assessed by using an IL-6 dependent murine hybridoma cell line. MH-60. BSF-2. In addition, we observed that pretreatment of SMC with IL-1 for 48 h stimulated growth of SMC during the 96 h incubations, as assessed by cell number (p less than 0.05). These results suggest that IL-1 may contribute to the pathogenesis of inflammatory and immunological vasculitis by the augmentation of IL-6 release and growth of SMC.     

Correlation of anti-cytoskeleton antibody activities in synovial fluid with interleukin-6 in patients with osteoarthritis and inflammatory joint disease

Summary Synovial fluids and sera from patients with rheumatoid arthritis, psoriatic arthritis, yersinia arthritis, Behçet's syndrome, Crohn's disease, and osteoarthritis were tested for antinuclear antibodies and antibodies to five cytoskeletal components in sensitive enzyme-linked immunosorbent assay (ELISA) systems and for IL-6 concentrations in a proliferation assay (IL-6 dependent hybridoma cell line B13.29, subclone B9). Statistically significant correlations between antibody activities and IL-6 levels were found for vimentin antibodies (r= 0.56; p<0.05) and actin antibodies (r= 0.44;p<0.05). In patients with chronic and active disease like rheumatoid arthritis and psoriatic arthritis, optical densities measured by vimentin- and actin-ELISA were significantly different from those measured in patients with osteoarthritis. To date only a few reports exist concerning the incidence of antibodies in synovial fluids. We have shown to our knowledge for the first time that IL-6 seems to induce synovial fluid antibody activities restricted to cytoskeletal components of synoviocytes (i.e., vimentin and actin). Synovial fluid antibody activities against vimentin and actin appear to be markers of activity in patients with inflammatory joint disease.Abbreviations ELISA enzyme-linked immunosorbent assay - ANA antinuclear antibodies - ACA antibodies against cytoplasmic components - RA rheumatoid arthritis - OA osteoarthritis - SF synovial fluid - RF rheumatoid factors - FCS fetal calf serum - IFT immunofluorescence microscopy - PBS phosphate buffered saline - BSA bovine serum albumin - OD optical density - SLE systemic lupus erythematosus - IL-6 interleukin-6 - HGF hybridoma growth factor - BSF-2 B-cell-stimulatory factor - DMEM Dulbecco's modified Eagles Medium     

条件培养液中杂交瘤生长因子对人—鼠杂交瘤生长、克隆化及抗体分泌的影响

本文报道了含有杂交瘤生长因子的人纤维母细胞系CRL1506和EB病毒转化经克隆化的人B淋巴母细胞系N23两种条件培养液,对分泌肾综合征出血热病毒人单克隆抗体的人-鼠杂交瘤和人—(人—鼠)杂交瘤细胞系的生长、克隆化及抗体分泌有明显促进作用,并排除了白细胞介素2和白细胞介素4作用的可能性。结合文献报道,初步认为来自这两种条件培养液的杂交瘤生长因子很可能含有白细胞介素6。正确选择促进人—鼠杂交瘤生长、克隆化和抗体分泌的条件培养液,对于克服生产人单克隆抗体杂交瘤技术所存在的融合率低、克隆化难、抗体产量少等困难具有较大的应用价值。     

Enhancement of polymorphonuclear leukocyte (PMN) function by OK-432

The influence of OK-432, a streptococcal preparation, on human polymorphonuclear leukocytes (PMN) was examined. OK-432 increased O₂⁻ generation by PMN in oral cancer patients, and sustained the production of O₂⁻ in patients on chemoradiotherapy. Enhanced O₂⁻ generation was also observed when PMN were cultured with 10⁻² KE/ml OK-432 for 1 h and then stimulated with phorbol myristate acetate or formyl-metionyl-leucil-phenylalanine (FMLP). In addition, PMN O₂⁻ generation was promoted by culture supernatants of peripheral blood mononuclear cells (PBMC) incubated with 10⁻³ or 10⁻² KE/ml OK-432. Furthermore, OK-432 (10⁻³−10⁻² KE/ml) enhanced the chemiluminescence of FMLP- and PMA-stimulated PMN. However, nitroblue tetrazolium reduction and myeloperoxidase activity were only minimally enhanced. Not only the candidacidal activity of PMN but also antibody-dependent cell-mediated cytotoxicity against Candida and Raji cells were enhanced in correspondence with the increased generation of reactive oxygen species.Culture of PMN or PBMC for 24 h with OK-432 resulted in a concentration-dependent increase in the substantial production of interleukin-1β, interleukin-6 and tumor necrosis factor-α. OK-432 also enhanced granulocyte-macrophage colony stimulating factor and gamma-interferon generation by leukocytes in a dose-dependent manner. Our research indicates that OK-432 enhances PMN function directly as well as via the promotion of cytokine production, and suggests that these effects of OK-432 could be beneficial in immunosuppressed patients.     

Analysis of antitumor effects of OK-432 against syngeneic mouse fibrosarcoma: combination effect of OK-432 and recombinant lymphokines

OK-432, a streptococcal preparation with potent biological response modifying activities, had the ability to cure mice bearing BAMC-1 (fibrosarcoma) ascites when it was injected intraperitoneally five times, 2, 4, 6, 8, and 10 days after the tumor inoculation. Previously, it was shown that the OK-432 injection on day 2 was indispensable since only a minimal antitumor effect was obtained when an inflammation-inducing agent such as thioglycolate instead of OK-432 was injected on day 2, followed by four OK-432 injections on days 4, 6, 8 and 10. In the present study, the injection of OK-432 on day 2 and a subsequent injection of either IL-2 or IFN-gamma on day 4 or 6 showed a significant antitumor effect, achieving a complete cure in approximately 50% of mice treated, although none of the mice could be cured by a single injection of either OK-432, IL-2, or IFN-gamma on day 2. Interestingly, however, the mice treated with an injection of a lymphokine (IL-2 or IFN-gamma) on day 2 followed by OK-432 on day 4 were not cured either. Peritoneal cells on day 12 in mice treated with OK-432 and either of the lymphokines contained pantropic killer cells, which were Thy-1+ and asialo GM1+ (AsGM1+). Moreover, the antitumor effect of the combined treatment was abolished when mice were pre-treated with anti-AsGM1. No significant antitumor effect was observed in athymic nu/nu mice. Together with our previous findings, these results indicate that lymphokines induced by OK-432 administration may account for some of its therapeutic effects and that these lymphokines may also facilitate the subsequent induction of specific killer cells. These results warrant further investigation on possible effective therapeutic protocols with the combined use of OK-432 and lymphokines.     

Production of cytotoxic factor into mouse peritoneal fluid by OK-432, a streptococcal preparation

A cytotoxic factor was induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with a streptococcal antitumor preparation, OK-432. No cytotoxic effect on L-929 cells was observed in the peritoneal fluids of mice singly treated with OK-432 or LPS. Various mouse and human tumor cell lines were effectively killed by this peritoneal cytotoxic factor, though normal cell lines were insensitive, which indicates that this factor is not species-specific. The highest level of cytotoxic activity was obtained when LPS was given to mice 5 days after the injection of OK-432. The optimal time for collection of peritoneal fluids for the cytotoxic factor was 2 h following the LPS injection. Interferon activity was found to be negative by the plaque reduction test using L-929 cells with vesicular stomatitis virus. These results suggest that this cytotoxic factor is similar to the tumor necrosis factor (TNF) in the mouse serum.     

In vitro augmentation of natural killing activity by OK-432

OK-432, a streptococcal preparation, augmented the natural killing (NK) activity of peripheral blood lymphocytes of normal donors and cancer patients against both NK sensitive and resistant human target cells in vitro. The enhancement of NK activity was evident after 4 h pretreatment and maximum by 16-24 h. The manifestation of OK-432 induced augmentation required active cell metabolism, RNA and protein synthesis but no DNA synthesis of lymphocytes. The supernatants produced by OK-432 stimulated lymphocyte cultures had no enhancing substance nor interferon. Anti-interferon antibodies did not inhibit boosting activity of OK-432. Large granular lymphocytes were involved in both spontaneous and OK-432 induced cytotoxic activity. The proportion of lymphocytes conjugating to target cells was not changed by OK-432. These results suggest that OK-432 augments cytotoxic activity of large granular lymphocytes having ability to recognize target cells independent of interferon induction.     

B细胞生长因子活性上清对人—人杂交瘤细胞生长影响的研究

本研究用金黄色葡萄球菌(Cowan 1株)死菌制剂(SAC)刺激人B淋巴细胞建立了人B细胞生长因子(BCGF)的检测系统。发现经PHA刺激的人扁桃体细胞培养上清可以促进SAC活化的B细胞的生长,~3H-TdR掺入率提高了2～3倍。同时,用~3H-TdR掺入法和有限稀释法观察了该上清对人-人B细胞杂交瘤生长和克隆率的影响,发现其可以促进人-人B细胞杂交瘤的生长、~3H-TdR掺入率提高2～3倍,可提高克隆率3倍以上。但人-人B细胞杂交瘤可吸收掉上述BCGF活性上清中的促杂交瘤细胞生长因子(HGF)活性,而对BCGF活性无明显影响。提示该上清同时具有人BCGF和HGF活性,部分BCGF活性与HGF活性可能是由不问的分子所介导。