Effects of combined antigrowth factor receptor treatment on in vitro growth of multiple myeloma.

BACKGROUND: Although passive serotherapy for cancer with monoclonal antibodies is an attractive concept, it has unfortunately had limited efficacy in clinical trials. An alternative approach to passive serotherapy is targeting cell surface growth factor receptors with monoclonal antibodies. With some limitations, anti-growth factor receptor antibodies can limit cell growth by blocking stimulatory or trophic growth factor receptors and by marshaling in vivo antitumor immune responses. PURPOSE: The purpose of our study was to determine the extent to which anti-interleukin-6 (IL-6) and anti-transferrin (Tf) receptor antibodies, when used individually or combined, could limit myeloma cell growth. METHODS: The four myeloma cell lines studied varied in IL-6 responses from factor independence (myeloma cell lines 8226 and U266) to strict factor dependence (OCI-My4 myeloma cells and human acute myelogenous leukemia [AML] cell line UCSD/AML1). IL-6 RNA was detected using reverse transcriptase-polymerase chain reaction. IL-6 protein was detected in U266 supernatant by growth stimulation of UCSD/AML1 cells and by enzyme-linked immunosorbent assay. For cell growth assays, cell lines were plated with various concentrations of IL-6 and anti-receptor antibodies and [3H]thymidine uptake determined after 3 days. Cells were grown in varying concentrations of IgG1 monoclonal anti-Tf receptor antibodies E2.3 and A27.15 or antibodies PM1, AUK 146-15, AUK 64-7, or AUK 12-20 to the human IL-6 receptor-alpha protein. Tf and IL-6 receptors were detected by immunofluorescence staining. RESULTS: Using short-term proliferation assays, anti-Tf receptors and anti-IL-6 antibodies caused dose-dependent growth inhibition of varying degrees, and, in one of three cell lines, a combination of anti-Tf and anti-IL-6 antibodies showed supra-additive growth inhibition. IL-6-independent cells were inhibited by anti-Tf receptor antibodies, while IL-6-dependent cells were resistant to these antibodies but sensitive to anti-IL-6 receptor. Factor-dependent myeloma cells exposed to either anti-Tf or anti-IL-6 receptor antibodies for 48 hours lost colony-forming capability. A combination of anti-Tf and anti-IL-6 antibodies increased elimination of colony-forming cells at 24 hours. CONCLUSIONS: Anti-receptor antibodies have distinct patterns of myeloma cell growth inhibition and inhibit in vitro growth of factor-dependent myeloma cells. Combinations of anti-growth factor receptor antibodies also increase toxicity for IL-6-dependent myeloma colony-forming units.

Sensitization of human renal cell carcinoma cells to cis-diamminedichloroplatinum(II) by anti-interleukin 6 monoclonal antibody or anti-interleukin 6 receptor monoclonal antibody.

Cytotoxic chemotherapy has shown little antitumor activity against renal cell carcinoma (RCC). It has been demonstrated that RCC cells secrete interleukin 6 (IL-6) and express IL-6 receptors (IL-6Rs). IL-6 inhibits apoptosis and enhances manganese superoxide dismutase expression. Several anticancer chemotherapeutic agents exert their cytotoxic activity in part through the induction of apoptosis and the production of free radicals. Thus, the resistance of RCC cells to the anticancer agents might correlate with IL-6 expression. The present study tested this hypothesis by examining the effect of anti-IL-6 mAb and anti-IL-6R mAb on the sensitivity of human RCC cells to anticancer chemotherapeutic agents. Treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb in combination with cis-diamminedichloroplatinum(II) (CDDP) or mitomycin C overcame their resistance to CDDP or mitomycin C. However, treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb in combination with Adriamycin, vinblastine or 5-fluorouracil did not overcome their resistance to these anticancer agents. Treatment of CDDP-resistant Caki-1 cells (Caki-1/DDP), two other RCC cell lines (ACHN and A704), and three freshly derived RCC cells with CDDP in combination with anti-IL-6 mAb or anti-IL-6R mAb reversed the resistance to CDDP in all these tumors. We then studied the effectiveness of other platinum derivatives. Treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb enhanced their sensitivity to carboplatin, but not to trans-diamminedichloroplatinum(II). Several experiments investigated the mechanism of the antibody-mediated sensitization of RCC cells to CDDP. Incubation of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb did not change the intracellular accumulation of CDDP. The expressions of the multidrug resistant phenotype (gp170) and c-myc oncogene were not affected by the antibody-mediated sensitization. Treatment of Caki-1 cells with the anti-IL-6 mAb or anti-IL-6R mAb down-regulated the expression of glutathione S-transferase pi mRNA. This study demonstrates that treatment of RCC cells with CDDP in combination with anti-IL-6 mAb or anti-IL-6R mAb can overcome their CDDP-resistance and that the down-regulation of glutathione S-transferase pi expression by anti-IL-6 mAb or anti-IL-6R mAb might play a role in the enhanced cytotoxicity obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Reshaping a human antibody to inhibit the interleukin 6-dependent tumor cell growth.

The mouse PM-1 monoclonal antibody binds to the human interleukin 6 receptor, inhibits IL-6 functions, and shows strong antitumor cell activity against multiple myeloma cells. In order to be effective as a therapeutic agent administered to human patients in repeated doses, reshaped human PM-1 antibodies consisting of human REI-based light chain and NEW-based heavy chain variable regions were designed and constructed with the assistance of a structural model of the mouse PM-1 variable regions. The best reshaped human PM-1 antibody is equivalent to mouse or chimeric PM-1 antibody in terms of antigen binding and growth inhibition against multiple myeloma cells. Only a few minor changes in the human framework regions were required to recreate the mouse PM-1 antigen-binding site within a human antibody. The reshaped human PM-1 antibody, therefore, could be efficacious in human multiple myeloma patients.

Establishment of a novel myeloma cell line KPMM2 carrying t(3;14)(q21;q32), which proliferates specifically in response to interleukin-6 through an autocrine mechanism.

We established a new human myeloma cell line, KPMM2, which proliferates specifically in response to IL-6 via an autocrine mechanism. The proliferative response of KPMM2 cells to exogenous IL-6 was significantly stimulated in a dose-dependent manner. The growth was markedly inhibited by an anti-IL-6 mAb and an anti-IL-6 receptor (IL-6R) mAb in a dose-dependent manner. KPMM2 cells expressed IL-6 and IL-6R mRNA by RT-PCR. Flow cytometric analysis showed cell surface expression of IL-6R. IL-6 protein was detected in the culture supernatant by ELISA. IL-11, oncostatin M and leukemia inhibitory factor had no effect on the proliferation of KPMM2 cells although interferon-alpha and interferon-gamma inhibited the growth. Furthermore, KPMM2 cells bore a t(3;14)(q21;q32) translocation and this finding is of potential interest for future studies in the light of the nuclear protein BM28 (CDCL1, for cdc-like 1) mapped on 3q21, which plays an important role in the cell cycle. In this report, we demonstrated completely an IL-6-dependent autocrine growth mechanism in KPMM2 cell line. This cell line may be useful to investigate the pathogenesis of multiple myeloma and to evaluate the therapeutic potential of IL-6 blocking agents in vitro and in vivo.

Effects of all-trans retinoic acid and antireceptor antibodies on growth and programmed cell death of human myeloma cells.

In contrast to cytotoxic agents inducing rapid cell death, biological agents such as hormones, vitamins (e.g., retinoids), cytokines, and antireceptor antibodies act slowly and may alter ratios between cell growth and programmed cell death (apoptosis). We showed previously that anti-interleukin 6 (IL-6) and antitransferrin (Tf) receptor antibodies inhibited in vitro growth and induced death of myeloma cells. Retinoids also inhibit in vitro growth of human cancer cells and decrease IL-6 receptor display and autosecretion by some myeloma cells. Retinoids may also antagonize in vitro growth-promoting effects of iron and transferrin. To develop a novel strategy for treating myeloma, we examined antiproliferative and cytotoxic effects of retinoids in combination with anti-Tf or anti-IL-6 receptor antibodies. Myeloma cell lines were cultured with retinoids with or without anti-growth factor receptor monoclonal antibodies. Both all-trans retinoic acid (ATRA) and 13-cis-retinoic acid showed variable, dose-dependent inhibition of myeloma cell line growth. ATRA also induced significant down-regulation of myeloma IL-6 receptors and inhibited IL-6 autosecretion by myeloma cells. Antiproliferative effects of ATRA were increased by coculture with anti-Tf but not anti-IL-6 receptor antibodies. Colony-forming assays showed that antiproliferative effects of anti-Tf receptor antibodies were largely reversible, but 1 microM ATRA was cytotoxic to myeloma cells. To assess apoptosis, a flow cytometry assay detecting DNA damage was used. Using previously studied cell line models, flow cytometry detected programmed cell death induced by transforming growth factor beta1 in leukemia cells and by anti-growth factor receptor antibody treatment of IL-6-dependent myeloma cells, treatments which caused only modest increases in the percentage of cells undergoing morphological apoptosis and increased internucleosomal DNA degradation. Flow cytometry analysis of ATRA and anti-Tf antibody-treated myeloma cells also showed evidence for apoptosis induced by ATRA, but not with anti-Tf receptor antibodies. These changes were apparent several days before detection of internucleosomal DNA degradation on agarose gels in 8226 cells but were not detected at any time in U266 cells, which underwent cell death but showed no DNA damage using flow cytometry or degradation on agarose gels. Retinoids merit further study as possible maintenance or chemoprevention therapies for clonal plasma cell disorders and for treating paraneoplastic disorders such as Castleman's disease. Flow cytometry rapidly detects apoptosis induced by biological agents and may be useful for in vitro screening of novel biological therapies.

Humanization of a mouse anti-human interleukin-6 receptor antibody comparing two methods for selecting human framework regions.

Mouse monoclonal antibody AUK12-20 binds to human IL-6 receptor and inhibits IL-6 functions. It has been humanized by CDR-grafting for therapeutic use. In the design of reshaped human AUK12-20 VL region, the human framework regions (FRs) from the human Bence-Jones protein REI were used. The reshaped human AUK12-20 light chain, in combination with chimeric AUK12-20 heavy chain, bound to antigen as well as chimeric AUK12-20 antibody. In the design of reshaped human AUK12-20 VH region, two sets of the human FRs were chosen and compared. One set was from the consensus amino acid sequence for human VH regions subgroup (HSG)-I and the other set was from human antibody HAX, the most similar human VH region found in a database of human immunoglobulin sequences. The HSG-I-based and the HAX-based reshaped human AUK12-20 heavy chains in combination with the reshaped human AUK12-20 light chain, showed approximately 90 and 100% antigen-binding and competition-binding activities as compared to the chimeric or mouse AUK12-20 heavy chains. Most importantly, these humanized antibodies inhibited the IL-6-dependent tumor cell growth as well as the original mouse antibody suggesting that these humanized antibodies could be efficacious in human patients. Our results show that both approaches for the design of reshaped human antibodies can be used for successful humanization. The approach based on FRs from the most similar individual human antibody, however, seemed to be best for designing a reshaped human antibody that mimicked as closely as possible the original mouse antibody.

The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity.

A mouse monoclonal antibody, anti-HM1.24 (IgG2a/kappa), binds to a surface antigen preferentially overexpressed on multiple myeloma (MM) cells, and exhibits potent antitumor cell activity against MM cells by antibody-dependent cell-mediated cytotoxicity (ADCC). To develop an antibody-based immunotherapy against MM, a humanized anti-HM1.24 antibody, in which all FRs correspond to naturally processed human FRs, has been successfully constructed with the aid of both the hybrid variable region and two-step design methods. This humanized anti-HM1.24 antibody (IgG1/kappa) is able to effectively induce ADCC against human myeloma KPMM2 and ARH77 cells in the presence of human PBMCs as effectively as a chimeric anti-HM1.24 antibody. The humanized anti-HM1.24 antibody, therefore, could be expected as a potent immunotherapeutic agent for MM patients.

Human erythropoietin stimulates murine megakaryopoiesis in serum-free culture.

Recombinant erythropoietin (Epo) was capable of stimulating murine megakaryopoiesis both in serum-containing and serum-free cultures, although a relatively high amount of Epo was necessary to provide sufficient stimulus for colony growth. This observation was further confirmed by experiments using nonadherent, nonphagocytic, and T-cell-depleted marrow cells in which Epo stimulated the growth of single megakaryocytes, as well as clusters or colonies. Total plate analysis revealed that twice as many single megakaryocytes and two-cell aggregates were generated by Epo than generated by pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). The number of colonies with four or more cells formed by PWM-SCM, however, was significantly higher than that generated by Epo. These results suggest that in comparison to the factor(s) in PWM-SCM, Epo stimulates the growth of more mature progenitors.

Acquisition of growth autonomy and tumorigenicity by an interleukin 6-dependent human myeloma cell line transfected with interleukin 6 cDNA.

In human multiple myeloma, an autocrine growth mechanism through interleukin 6 (IL-6) has been advocated. However, growth of myeloma cells in vitro is poor except for established cell lines, and IL-6 autocrine growth is quite rare in myeloma cell lines. In the present study, we devised a model of IL-6 autocrine growth in vitro by transfecting IL-6 cDNA into a human myeloma cell line that had a proliferative response to IL-6 but did not produce IL-6. After IL-6 transfection, the cells proliferated in culture media without IL-6, and their growth rate was elevated at higher cell densities. IL-6 was detected by enzyme-linked immunosorbent assay in the culture media of the transfectants. IL-6 mRNA was distinctly expressed in these cells when analyzed by Northern blotting. The growth of the transfectants was definitely inhibited by anti-IL-6 or anti-IL-6 receptor monoclonal antibodies. Furthermore, the transfectants were successfully transplanted to nude mice. These results indicate that the myeloma cells obtained growth autonomy in vitro through IL-6 and tumorigenicity in vivo, after IL-6 transfection.

Endogenous bone-resorbing factors in estrogen deficiency: cooperative effects of IL-1 and IL-6.

Estrogen deficiency causes a marked bone loss by stimulating osteoclastic bone resorption. To explore the endogenous bone-resorbing factors involved in estrogen deficiency, we examined the bone-resorbing activity present in the supernatant fraction of mouse bone marrow collected from ovariectomized (OVX) mice. Adding bone marrow supernatants at 20-80% to organ cultures of mouse long bones dose-dependently stimulated bone resorption. The endogenous bone-resorbing activity present in bone marrow supernatants from OVX mice was much higher than that from sham-operated mice 2-4 weeks after surgery, and it was significantly diminished by indomethacin in vitro. Anti-IL-1 alpha antibody completely neutralized the bone-resorbing activity present in bone marrow supernatants from OVX mice. Antibodies against IL-1 beta, IL-6, and IL-6 receptors also neutralized it, but partially. The concentration of IL-1 alpha measured by ELISA was much higher in bone marrow supernatants than in sera, but it was not appreciably changed before or after OVX. The concentration of IL-1 beta in bone marrow supernatants from OVX mice was less than the detection limit. OVX stimulated IL-1 activity in bone marrow supernatants measured by means of the proliferation of thymocytes. However, the level of IL-1 alpha present in bone marrow supernatants from OVX mice was insufficient to stimulate bone resorption. Compared with the serum concentration, bone marrow supernatants contained a much higher level of IL-6 as well, and it was further increased by OVX. However, IL-6 alone present in bone marrow supernatants from OVX mice again did not stimulate bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional induction of cyclooxygenase-2 in osteoblasts is involved in interleukin-6-induced osteoclast formation.

Interleukin-6 (IL-6) induces osteoclast-like cell (osteoclast) formation in a dose-dependent fashion in cocultures of mouse bone marrow cells and osteoblastic cells when soluble IL-6 receptor (sIL-6R) is present. Simultaneous treatment with submaximal doses of IL-1alpha and IL-6 with sIL-6R caused marked induction of osteoclast formation and PGE2 synthesis. These effects were suppressed by adding neutralizing antibodies against IL-1alpha or IL-6R and were totally abolished by adding nonsteroidal antiinflammatory drugs, such as indomethacin and a selective cyclooxygenase-2 (COX-2) inhibitor (NS398). In mouse osteoblastic cells, both IL-1alpha and IL-6 with sIL-6R markedly induced messenger RNA expression of COX-2, but not COX-1, as determined by Northern blot analysis, and luciferase activity in cells stably transfected with a COX-2 promoter-luciferase fusion construct. IL-6 and sIL-6R, when added separately, did not stimulate COX-2 messenger RNA expression. Simultaneous addition of IL-1alpha and IL-6 with sIL-6R to osteoblast cultures cooperatively induced transcription of COX-2, which was associated with a marked increase in COX activity measured by the conversion of arachidonic acid into PGE2. The increased PGE2 synthesis by osteoblasts may play an important role in osteoclastogenesis induced by submaximal doses of IL-1 and IL-6.

Soluble interleukin-6 (IL-6) receptor in the sera of pregnant women forms a complex with IL-6 and augments human chorionic gonadotropin production by normal human trophoblasts through binding to the IL-6 signal transducer.

To study the role of soluble interleukin-6 receptor (sIL-6R) during pregnancy, sIL-6R levels in the sera of pregnant women in the first, second, and third trimesters were determined and found to remain unchanged during pregnancy, but were significantly higher than those in nonpregnant women in the follicular, ovulatory, and luteal phases of the menstrual cycle (P < 0.001). IL-6 levels, however, in the sera of pregnant women at all trimesters showed no difference from those in nonpregnant women at any stage of the menstrual cycle. Recombinant sIL-6R (rsIL-6R) augmented hCG production by rIL-6-stimulated trophoblasts dose dependently, but failed to enhance hCG production by unstimulated trophoblasts. rIL-6- and rsIL-6R-induced hCG production was significantly blocked by anti-IL-6R antibody, PM1; antisignal transducing glycoprotein 130 (gp130) antibody, GPX7; or a tyrosine kinase inhibitor, genistein. Thus, sIL-6R in serum from pregnant women forms a complex with placental and decidual IL-6 in a manner similar to trophoblast membrane-bound IL-6R. These two discrete types of IL-6R and IL-6 complex might act cooperatively by binding to gp130 and subsequently evoking tyrosine kinase activity in the trophoblasts to produce hCG in vivo.

The influence of interleukin-6 on the growth of human esophageal cancer cell lines.

We reported that human esophageal cancer cell lines (ECC) (YES-1, -2, -3, -4, -5, and -6) produced interleukin-6 (IL-6). We, therefore, investigated the growth effects ([3H]thymidine uptake assay and direct cell count) of IL-6 on these ECC. IL-6 receptor (R) and GP-130 mRNA were detected in all the ECC, using reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and IL-6R was detected in one (YES-3) by immunohistochemical staining. IL-6, anti-IL-6 monoclonal antibody (mAb), or anti-IL-6R mAb caused no reproducible enhancement or suppression of [3H]thymidine uptake by all six ECC. Direct cell count also revealed that the growth enhancement or suppression by IL-6, anti-IL-6 mAb, or anti-IL-6R mAb was relatively small. Particularly, there was no significant sensitivity of YES-3 cells, which definitely produce IL-6 and express IL-6R for IL-6, anti-IL-6 mAb, or anti-IL6R mAb. These results suggest that some esophageal cancers may produce IL-6 and express IL-6R. However, no major interactions between IL-6 and the growth of human esophageal cancer cell lines were detected in this study.

Production of interleukin 6 by human spleen cells stimulated with streptococcal preparation OK-432.

The streptococcal preparation OK-432 was tested for the ability to stimulate human spleen leukocytes (SPL) for generation of interleukin 6 (IL-6). When SPL were cultured with OK-432 for 24 h in serum-free T medium, the cell-free supernatant induced production of IgM in the SKW6.CL-4 and IgG in the CESS human B cell line, while no such activity was detected in unstimulated SPL culture. The activity was neutralized by treatment with antiserum directed against B cell stimulatory factor 2 (BSF-2). An optimum production of BSF-2 was observed when SPL were stimulated with 10 micrograms/ml of OK-432. The culture supernatant also induced proliferation of IL-6-dependent murine hybridoma MH-60.BSF2 (hybridoma growth factor; HGF). It is thus evident that the molecule produced by OK-432-activated human SPL is BSF-2/HGF/IL-6. These results indicate that the antitumor agent OK-432 stimulates human spleen cells to produce IL-6.

Estimation of interleukin 6 production by reverse transcriptase-polymerase chain reaction in four human myeloma cell lines.

Although an autocrine growth mechanism through interleukin 6 has been advocated in human myeloma cells, reports of IL-6 production by cells from established myeloma cell lines are rare. In the present study, we examined whether or not a minute amount of interleukin 6 is produced in 4 human myeloma cell lines. IL-6 production was not detected in any of the 4 lines by enzyme immunoassay, bioassay with two interleukin 6-dependent murine hybridoma cell lines and Northern hybridization. However, we detected interleukin 6 mRNA in one (U266) of the 4 lines by the reverse transcriptase-polymerase chain reaction. Nevertheless, the proliferation of all 4 lines was not inhibited by an anti-interleukin 6 antibody. These results suggest that autocrine stimulation by interleukin 6 is not involved in the majority of human myeloma cell lines.

Purification and characterization of soluble human IL-6 receptor expressed in CHO cells.

An immunosorbent assay system to detect genetically engineered IL-6 receptor (IL-6R) was established, whereby soluble IL-6 receptor (sIL-6R) was detected in the culture medium when sIL-6R cDNA was transfected into COS1 cells. A stably transformed Chinese hamster ovary (CHO) cell line constitutively expressing sIL-6R has been established. The recombinant sIL-6R was purified to homogeneity by sequential filtration and chromatography of the culture medium. The recombinant sIL-6R augmented the sensitivity of M1 cells to IL-6 in growth inhibition assay in a dose-dependent manner. Furthermore, a radioisotope immunosorbent assay (RIA) utilizing recombinant sIL-6R was established which could detect IL-6 in a quantity as low as 10 ng/ml.

Therapeutic potential of humanized anti-interleukin-6 receptor antibody: antitumor activity in xenograft model of multiple myeloma.

A xenograft model of human multiple myeloma (MM) was established in athymic nude mice using S6B45 cells whose growth is dependent on IL-6 in an autocrine fashion. S6B45 cells were inoculated s.c. into mice pretreated with 500 cGy X-ray and anti-asialo GM1 antibody. In more than 90% of the mice, a palpable tumor emerged within 30 days at the inoculation site. Histological observation of the tumor section revealed that the tumor mass was composed of two different phenotypes of myeloma cells, corresponding to plasmablasts and mature plasma cells. I.v. injection of more than 0.125 mg of mouse monoclonal antibody (PM1) against human IL-6R (hIL-6R) on days 1, 3 and 5 markedly delayed the time of tumor incidence. One mg of anti-hIL-6 antibody (MH166) also strongly inhibited the growth of S6B45, whereas control antibody (MOPC31C) and anti-hIL-6R antibody without neutralizing activity (AUK181-6) produced no significant effects. To reduce the antigenicity of PM1 in human, mouse-human chimeric PM1 (chPM1) with human IgG1 constant region and humanized PM1 (hPM1), human IgG1 with mouse complimentarity determining regions, were constructed and evaluated for their in vivo antitumor activity in our model. The in vivo efficacy of these recombinant antibodies (chPM1 and hPM1) was shown to be equivalent to that of the original PM1. These results indicate that the antitumor activity of PM1 is completely recreated in hPM1, and that blocking of the IL-6 signal by this humanized antibody could be a potent therapy for MM.

Interleukin 6 inhibits delayed-type hypersensitivity and the development of adjuvant arthritis.

We studied the effect of interleukin 6 (IL 6) on the delayed-type hypersensitivity (DTH). In mice immunized with sheep red blood cells (SRBC), a DTH response was evoked by antigen challenge into the hind paw 5 days after immunization. The magnitude of the response was assessed by footpad swelling measured 24 h after antigen challenge. IL 6 significantly suppressed the DTH in its induction phase in a dose-dependent manner when administered s.c. into the back at a dose of greater than 2.5 micrograms twice a day (5 micrograms/day) for 5 consecutive days from the day of immunization (day 0) to 1 day before antigen challenge (day 4). Heat-inactivated IL 6 did not suppress the DTH response. Furthermore, the suppressive activity of IL 6 was completely abolished by affinity chromatography on an anti-IL 6 antibody. This suppression was also obtained when IL 6 was administered only on day 0 and day 1, but not on days 3 and 4. This indicates that IL 6 acts on the early part of the induction phase of DTH development. Furthermore, footpad swelling was suppressed even by the administration of IL 6 after antigen challenge. These results show that IL 6 suppresses both the induction and effector phases of DTH. To confirm further this inhibitory effect of IL 6, we examined its effect on the development of adjuvant arthritis in rats. Administration of IL 6 also significantly suppressed the development of adjuvant arthritis.

Enhanced hematopoiesis in vivo and in vitro by splenic stromal cells derived from the mouse with recombinant granulocyte colony-stimulating factor.

Splenic stromal cells (CF-1 cells) were established from a mouse administered recombinant human granulocyte colony-stimulating factor (rG-CSF) to clarify the mechanism of splenic extramedullary hematopoiesis induced by the factor. The cells were negative for alkaline phosphatase, factor VIII-related antigen, mac I, and phagocytosis. They were positive for acid phosphatase, collagen type I, collagen type III, and fibronectin. CF-1 cells were not converted to adipocytes in a confluent culture with 10(-6) mol/L hydrocortisone. [35S]rG-CSF bound to CF-1 cells specifically in the growth phase but not in the resting phase. The CF-1 cells had greater colony-stimulating activities than the normal splenic stromal cells. When CF-1 cells were added to bone marrow cells in the spleen colony-forming cells (CFU-S) assay, the number of colonies in the spleen increased between 1.4 and 1.8 times the control without these stromal cells. On the other hand, the normal splenic stromal cells had no effect on increasing the number of CFU-S colonies. Therefore, these data suggest that a factor-dependent hematopoietic microenvironment is generated in the spleen by rG-CSF, and the stromal cells that have the hematopoietic potency become dominant in splenic extramedullary hematopoiesis induced by rG-CSF.

Murine anti-human IL-6 monoclonal antibody prolongs the half-life in circulating blood and thus prolongs the bioactivity of human IL-6 in mice.

Human interleukin-6 (hIL-6) injected into mice increases antigen-specific antibody production. In the present study, we examined the possibility that anti-hIL-6 murine monoclonal antibody (MH-166) could neutralize this hIL-6 activity in vivo. Although MH166 completely neutralized hIL-6 activity in vitro, treatment in vivo with hIL-6 and MH166 combined unexpectedly increased both anti-dinitrophenyl (DNP) and anti-sheep red blood cell (SRBC) antibody production more than treatment with IL-6 alone did. To explain this phenomenon, the serum hIL-6 level was monitored following the MH166 administration. The hIL-6 level was significantly higher in the mice treated with both hIL-6 and MH166 than in the mice treated with hIL-6 alone during the 24 hr following hIL-6 administration. These results indicate that MH166 prolongs half-life of a xenogeneic hIL-6.