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Lentinan, an antitumor polysaccharide, has been assessed for its potential in vivo to augment erythroid progenitor cells and protect them from the cytotoxic effects of antitumor chemotherapeutics. Lentinan augmented the level of burst-forming units-erythroid (BFU-E) and accelerated the recovery of the reduced number of BFU-E in mice treated with 5-fluorouracil (5-FU); lentinan did not influence red blood cell counts or colony-forming unit-erythroid (CFU-E) numbers in the femoral marrow. A significant decrease in stem cell inhibitory factor (SCIF) activities in bone marrow and an increase in colony-forming unit-spleen (CFU-S) formation were observed in the lentinan-treated mice. The mechanism of augmented BFU-E formation may be partly due to augmented production of stem cells, giving rise to both CFU-GM and BFU-E. Furthermore, when lentinan administration was followed by administration of erythropoietin (Epo) in 5-FU-treated mice, increases in femoral marrow and splenic CFU-E formation and augmentation of reticulocyte counts were observed beyond the level observed in mice treated with Epo alone. These results suggest that lentinan may augment the effects of Epo on erythropoiesis in the course of anemia and the decreased erythropoiesis in cancer patients receiving chemotherapy.

To determine whether resistance to chemoimmunotherapy is acquired during therapy, we investigated the effects of chemotherapeutic agents and anti-tumour polysaccharide, lentinan, on the progression of Rous sarcoma virus-induced S908.D2 fibrosarcomas. The chemoimmunotherapy was effective against the parental S908.D2-bearing mice. Nearly all the mice that were treated with cyclophosphamide (CY) and lentinan achieved complete tumour regression. Only a few of the mice that achieved complete regression of the primary tumours showed a recurrence of the tumour in regional lymph nodes. S908.D2-vp.1 was established from metastatic tumours that developed in the regional lymph nodes of parental S908.D2-bearing mice during therapy. S908.D2-vp.2-or vp.3 cells were sequentially derived in a similar way from S908.D2-vp.1-or-vp.2-bearing mice respectively, in which complete tumour regression at each primary site was achieved during therapy. These lines acquired resistance to CY and lentinan and also to 5-fluorouracil (5-FU)/5'-deoxy-5-fluorouracil and lentinan. No significant difference in either the sensitivity to 5-FU or 4-deoxycyclophosphamide in vitro or in the susceptibility to immune effector cells was observed between the parental and progressed lines (S908.D2-vp1 -vp3). There was an increase in the level of prostaglandin E2 (PGE2) in the progressed lines during repeated therapy (parental, 1171 pg ml(-1); vp.1, 2199 pg ml(-1); vp.2, 5500pg ml(-1); vp3, 16187 pg ml(-1)). There was no significant increase in the production of transforming growth factor beta (TGF-beta). The amount of interleukin-2 (IL-2) produced by spleen cells isolated from the S908.D2-vp.2-bearing mice was decreased compared with the amount produced by the parental S908.D2- bearing mice. Furthermore, combination therapy with lentinan and IL-2 achieved complete tumour regression in all the mice transplanted with S908.D2 progressed tumour lines, although IL-2 alone did not show any anti-tumour effects in either the S908.D2 parental or progressed lines. The findings suggest that the reduced production of IL-2 induced an increase in the production of the PGE2 by progressed tumour lines is involved in the acquisition of resistance.

Effects of interleukin 6 (IL-6) on the functional capacity of the immune and hematopoietic systems in 5-fluorouracil (5-FU)-treated mice were determined. IL-6 (5 x 10(4) units/mouse/day) was administered s.c. for 7 days by implantation of an osmotic pump, since it was demonstrated that a much higher increase in the primary response to sheep RBC was observed by administration of slowly released rather than daily s.c. injection of IL-6. IL-6 perfusion significantly augmented anti-sheep RBC antibody responses depressed by 5-FU (150 mg/kg) treatment. IL-6 also was shown to stimulate hematological recovery in mice treated with 5-FU. Namely, IL-6 perfusion accelerated the recovery of the number of hematopoietic stem cells, granulocyte-macrophage progenitors, and mature neutrophils in the spleen, although IL-6 did not stimulate the recovery of the neutrophil count in blood. Recovery of the platelet count in blood was stimulated by IL-6. Furthermore, it was found that the endogenous IL-6 level in serum increased after 5-FU treatment, which suggests that IL-6 may play some role in the recovery of the immune and hematopoietic systems. Finally, we examined the effect of IL-6 on the survival of mice treated with a higher dosage of 5-FU (300 mg/kg). IL-6 perfusion produced a distinct increase in survival rate at Day 30 (74% versus 28%). It is of note that the number of bacteria (identified as Escherichia coli) cultured from the spleen and the liver decreased in IL-6-perfused mice. This IL-6-induced effect was accompanied by enhancement of an oxidative burst response. Moreover, the anti-E. coli antibody titer in serum was higher in IL-6-perfused mice than in control mice. These results suggest the possible use of IL-6 for stimulating the reconstitution of the immune and hematopoietic systems after chemotherapy treatment.

A murine B lymphoma cell line, WEHI-231, constitutively secreted a kind of B cell stimulatory factor (BSF) that induced proliferation and IgM secretion in splenic B cells as well as BCL1 cells. Growth- and differentiation-promoting activities were not separated by various kinds of chromatographies on the basis of the m.w., isoelectric point, or hydrophobicity, and the degree of both activities in crude supernatants, DEAE-Toyopearl, TSK-3,000SWG, Mono P, and Phenyl-5PW fractions increased in a dose-dependent manner with complete correlation. The partially purified factor (WEHI-231-BGDF) did not show any other activities, such as IL 1, IL 2, interferon, or colony stimulating factor. WEHI-231-BGDF induced proliferation and IgM secretion in activated B cells with low density, but not in small resting B cells. WEHI-231-BGDF showed synergistic effect with dextran sulfate but not with anti-Ig in the induction of proliferation or IgM secretion in small resting B cells. WEHI-231-BGDF did not show any effect on Xid B cells. The relationship with several T cell-derived BSF and the significance of B cell-derived BSF in the B cell responses are discussed.

The role of IL-6 in the antiproliferative effect of IL-1 for tumor cell lines was investigated using IL-1-sensitive cell lines. Human recombinant IL-1 alpha and IL-6 both inhibited the growth of an IL-1-sensitive cloned human melanoma cell line (A375-C6). However, IL-1 has greater maximum growth inhibitory activity than IL-6. Conditioned medium of the tumor cells that were treated with IL-1 contained IL-6 as determined by ELISA. Northern blot analysis revealed that IL-6 mRNA expression increased in IL-1-treated cells. In addition, antibody against human IL-6 neutralized about 50% of the antiproliferative effect of IL-1. The growth of an IL-1-resistant clone of A375 cells (A375-C5), which cannot be shown to express any detectable IL-1R, was inhibited by IL-6 to the same degree as A375-C6 cells. The A375-C5 cell line did not produce IL-6 or increase IL-6 mRNA after stimulation with IL-1. These results indicate that IL-6 mediates in part the antiproliferative effect of IL-1 on A375-C6 cells by acting as an autocrine antiproliferative factor. IL-1 also inhibited the growth of a malignant human mammary cell line (MDA-MB-415). IL-6 exhibited only slight growth inhibition in this cell line. Neither IL-6 production nor IL-6 mRNA expression was induced in this cell line by IL-1. Antibody against IL-6 did not neutralize the antiproliferative effect of IL-1. Therefore, for MDA-MB-415 cells IL-6 appeared not to be involved in the antiproliferative effect of IL-1. These results indicate that the antiproliferative effect of IL-1 involves at least two pathways, one IL-6 dependent and another IL-6 independent. The contribution of IL-6 to the antiproliferative effect of TNF was also examined. IL-6 appeared not to play a role in the antiproliferative effect of TNF in these cell lines.

The effects of human rIL-6/B cell stimulatory factor 2 (hrIL-6/BSF-2) from Escherichia coli on murine Ag, SRBC-specific antibody responses were examined in vitro and in vivo. HrBSF-2 was effective in augmenting the primary and the anamnestic plaque-forming cells response to SRBC in vitro. The augmentation of the primary response was apparent when B cell-enriched spleen cells (B cells) were cultured with BSF-2 in the presence of IL-2. On the other hand, hrBSF-2 alone strongly enhanced the anamnestic response in a dose-dependent manner when spleen cells from SRBC-immunized mice were used. These effects of BSF-2 were abolished completely by anti-BSF-2 antibody, but not by normal rabbit Ig. Cell depletion experiments indicated that L3T4 (CD4)+ T cells, but not Lyt-2(CD8)+ T cells, and adherent cells (macrophages) have an important role in this BSF-2-induced augmentation of the response. In addition, kinetic studies showed that hrBSF-2 acts on B cells in the anamnestic response even when added relatively late in the culture. Finally, it was determined whether BSF-2 also could be active in modulating antibody responses in vivo. BSF-2 was shown to enhance the primary and secondary antibody responses in mice. The most apparent effect of BSF-2 was observed in the secondary response.