G-CSF prevents the suppression of bone marrow hematopoiesis induced by IL-12 and augments its antitumor activity in a melanoma model in mice.

BACKGROUND: IL-12 has been successfully used in experimental tumor therapy. However, administration of this cytokine induces dose-dependent suppression of hematopoiesis that could potentially limit its use in clinical trials. We decided to examine whether the myelosuppressive activity of IL-12 could be corrected by the administration of G-CSF. MATERIALS AND METHODS: In the initial experiments the influence of IL-12 and/or G-CSF on bone marrow and spleen GM-CFC was evaluated. To examine whether C-CSF could influence the antitumor activity of IL-12 the combination therapy with these agents was carried out starting on day seven following inoculation of melanoma MmB16 cells into the footpads of B6D2F1 mice. To obtain insight into the mechanism of the observed augmented antitumor activity of the combination therapy with IL-12 and G-CSF, the influence of these cytokines on macrophage activity (cytotoxicity and nitric oxide release) was analyzed. RESULTS: In accord with our expectations, the application of G-CSF partially prevented the suppression of bone marrow myelopoiesis in IL-12 treated mice. Unexpectedly, G-CSF also showed potentiation of antitumor effects of IL-12 in this melanoma model. The augmented antitumor activity of combined IL-12/G-CSF immunotherapy could result from the enhanced stimulation of macrophage NO production and cytotoxicity. CONCLUSION: The simultaneous administration of IL-12 and G-CSF partially prevented suppression of bone marrow myelopoiesis in IL-12-treated mice. Moreover, treatment with these cytokines also results in potentiated antitumor effects in a murine melanoma model.

Serum levels of soluble tumor-necrosis-factor receptors in patients with benign and malignant HPV-associated anogenital lesions.

The levels of type-I and type-II soluble TNF-alpha receptors (sTNF-Rs) were evaluated in sera from patients with various human-papillomavirus-(HPV)-associated benign and malignant anogenital lesions using specific enzyme-linked immunobiological assays. In patients with benign HPV6/11-associated condylomata acuminata, the levels of sTNF-RI were significantly increased, while sTNF-RII were in normal range. Both types of sTNF-Rs were in normal range in patients with benign HPV16-associated grade-I/II and grade-III cervical intra-epithelial neoplasia. However, their levels were significantly increased in patients with HPV16/18-associated squamous cervical cancer and anogenital Bowen's carcinoma. Sera from patients with condylomata acuminata and anogenital carcinomas displayed significantly increased TNF-alpha-inhibitory activity, as revealed by L929 cell-cytotoxicity assay. Increased serum TNF-alpha-inhibitory activity correlated with higher levels of sTNF-Rs. Furthermore, this inhibitory activity could be specifically abrogated by htr9 and utr1 monoclonal antibodies recognizing TNF-RI and TNF-RII respectively. Our results strongly suggest that serum sTNF-Rs may protect tumor cells from cytotoxic/cytostatic effects of locally released TNF-alpha, and that elevated levels of circulating sTNF-Rs may facilitate the growth of HPV-associated anogenital lesions.

Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats.

Rat chondrocytes isolated from the articular-epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte-chondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.

Effect of immunosuppression on rejection of cartilage formed by transplanted allogeneic rib chondrocytes in mice.

The effect of short-term immunosuppressive treatment with antithymocyte serum-procarbazine (ATS-PCH) and cyclosporin-A (Cy-A) on survival of allogeneic rib chondrocyte grafts was examined morphologically and by evaluation of specific humoral and cellular antigraft immunity. The latter were evaluated by means of leukoagglutination and the indirect migration inhibition assay, respectively. Untreated recipients of syngeneic rib chondrocytes and untreated recipients of whole syngeneic and allogeneic rib cartilage served as controls. Transplanted syngenic rib chondrocytes formed cartilaginous nodules similar to rib cartilage in situ. These nodules contained hypertrophied chondrocytes, but neither physiologic resorption by vascularized connective tissue nor bone formation occurred after an observation period of longer than six weeks. Transplantation of allogeneic chondrocytes resulted in development of humoral and cellular antigraft immunity, and the formed cartilage was destroyed by infiltrating immune cells. Immunosuppression by ATS-PCH resulted in inhibition of graft destruction and a marked decrease of specific humoral antigraft immunity. Cellular antigraft immunity did not occur. Moreover, neither the histologic appearance of the cartilaginous nodules nor the results of immunologic response evaluations in the ATS-PCH-treated group differed from those in untreated whole allogeneic cartilage recipients. Treatment with Cy-A did not significantly improve survival cartilage formed by allogeneic chondrocytes.

Cartilage transplants in normal and preimmunized mice.

Syngeneic, H-Y incompatible and allogeneic rib and nasal cartilages from 5-day- and 8-week-old mice were transplanted into adult recipients. Some recipients of allogeneic transplants were preimmunized with splenocytes or chondrocytes from animals of the same strain as cartilage donors, and some with sheep red blood cells (SRBC). Syngeneic or allogeneic transplants independently of the age of the cartilage, did not evoke any detectable humoral or cellular response as judged by evaluation of the specific cytotoxic antibodies and indirect migration inhibition test, respectively. Allogeneic transplants of nasal cartilage were free of infiltrating lymphoid cells, but in the vicinity of rib cartilage grafts some lymphocytes were present. Animals preimmunized with allogeneic splenocytes or chondrocytes displayed both humoral and cellular response to donor cells. Allogeneic cartilage transplants from these animals were surrounded by heavy infiltrations. Preimmunization with SRBC did not evoke infiltrations around allogeneic cartilage grafts.

Comparison of bone formed in transplants of isolated scapular and vertebral osteoblasts.

To compare the properties of osteoblasts from various endochondrilia bones, scapular and calvarial osteoblasts were intramuscularly transplanted in "sandwiches" made of devitalized calvarial vaults. The structure of transplants produced by both types of bone cells appeared similar. In 4 week-old transplants woven bone with numerous osteoclasts predominated. The area occupied on the cross-sections of transplants by bone tissue was considerably larger than that of the bone marrow cavities. Transplants of 8-week-duration contained mainly cancellous bone, the number of osteoblasts was low and the area taken by medullary space was larger than that of bone tissue. This finding indicates that either osteoblasts from various endochondrlia bones have similar properties or that the possible differences in intrinsic features of these osteoblasts were masked by the conditions of transplantation.

Natural cell-mediated cytotoxicity against syngeneic rat chondrocytes originating from different types of cartilage.

Natural anti-chondrocyte cytotoxicity of normal rat splenocytes, peritoneal cells and thymocytes was tested by means of 51Cr-release assay. Chondrocytes derived from epiphyseal, costal, nasal, and auricular cartilages were used as target cells. In some experiments, erythroleukaemic K-562 cells, known as typical natural killer cell targets, were also used. All types of chondrocytes were lysed equally well by splenocytes. Peritoneal cells exerted a low cytotoxic effect, whilst very low, almost negligible, cytotoxicity was noted with thymocytes. Negative selection with antibodies and complement showed that spleen-derived anti-chondrocyte effector cells are endowed with surface ganglioside asialo-GM1. A similar result was obtained in parallel experiments with K-562 cells. Moreover, 'cold' target experiments demonstrated that the release of 51Cr from the labelled chondrocytes could be inhibited by addition of unlabelled chondrocytes and K-562 cells.

Difference in shape of bone formed by isolated calvarial and scapular osteoblasts transplanted under various conditions.

To study the influence of transplantation conditions on early stages of osteogenesis, isolated calvarial or scapular osteoblasts were injected into the leg or dorsal muscles (free transplants) or implanted after seeding on fragments of devitalized parietal bones (supported transplants) into dorsal muscles. The cross-sections of bone islands formed by calvarial osteoblasts in the different types of transplants were then compared according to their maximal breadth and length. Moreover, the same dimensions of pieces of bone formed by scapular osteoblasts in supported transplants were compared with those of bones formed in free transplants into leg muscles. Finally, comparison of the dimensions of cross-sections of supported transplants of calvarial and scapular osteoblasts was done. Calvarial osteoblasts in dorsal muscles produced a slightly higher percentage of wider and longer islands than those in leg muscles. In supported transplants of calvarial osteoblasts the percentage of narrow bone islands (breadth less than 100 microns) was considerably higher than in free transplants. Similarly, the percentage of narrow cross-sections in bones formed by scapular osteoblasts was higher in supported than in free transplants. In supported transplants of calvarial osteoblasts the percentage of narrow islands was higher than in similar transplants of scapular bone cells. It is suggested that the differences in shape of pieces of bone formed in supported and free transplants reflect the difference in mechanical conditions to which the bone cells were subjected. Furthermore, in supported transplants devitalized parietal bones could form a barrier for diffusion of nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of bone formed intramuscularly after transplantation of scapular and calvarial osteoblasts.

Previous work suggested that osteoblasts determine the size of the bone marrow area within the bone and that calvarial osteoblasts differ from those induced intramuscularly by cartilage formed by transplanted epiphyseal chondrocytes. This study reports morphological observations of bone formed by transplanted scapular and calvarial osteoblasts isolated from bones of young rats. In intact scapulas of 28-day-old rats the percentage area occupied by bone tissue in relation to bone marrow was 6 times larger than in parietal bones of comparable age. Isolated syngeneic scapular osteoblasts usually produced an ossicle with similar general structure and ratio bone tissue/bone marrow area as in intact scapulas. In transplants of calvarial osteoblasts numerous islands of bone tissue with a small amount of bone marrow appeared. Bone formed in allogenic transplants was rejected. These results suggest that osteoblasts from endochondral scapular bone may have different properties than those from intramembranous calvarial bones. Alternatively, the large amount of medullary space in bone produced by transplanted scapular osteoblasts could result from their contamination with bone marrow stromal cells.

Structural differences between bone formed intramuscularly following the transplantation of isolated calvarial bone cells or chondrocytes.

Bone formed in intramuscular transplants of isolated syngeneic calvarial bone cells in mice, was compared with endochondral bone induced by cartilage produced by analogous transplants of isolated epiphyseal chondrocytes, as well as with parietal bones forming the bulk of the calvaria. Transplanted calvarial cells produced islands of bone, some of which contained intraosseous cavities. Osteoclasts inside these cavities were observed only in 14-day-old transplants and bone marrow cells in 28-day and older transplants. On the contrary, bone marrow appeared soon after formation of bone trabeculae in endochondral bone. The percentage area occupied by bone marrow in these specimens was about twentyfold larger than in the bone formed by transplanted bone cells. On the other hand, the bone marrow area in the latter type of bone was somewhat smaller but of similar order as in parietal bones. Moreover, both in parietal bones and in bone formed by isolated bone cells, the bone marrow was devoid of fat cells which were numerous in bone arising by endochondral ossification. It appears, therefore, that the ratio of bone marrow to the bone tissue area in parietal bones depends more on the intrinsic properties of osteoblasts than on the local factors in the environment of the developing bone. In the case of bone induced by cartilage, the bone marrow/bone tissue area could be determined both by the extent of cartilage resorption by vascularized tissue and by the properties of osteoblasts.