Subunit structure of the high and low affinity human interleukin-15 receptors.

Radio-iodinated cytokines and monoclonal antibodies directed at the IL-2R beta- and gamma-chains were used to analyze the structure of the cell-surface IL-15 and IL-2 receptors expressed by the human lymphoma cell clone YT-2C2. YT-2C2 cells are IL-2R alpha negative and express IL-2R gamma (15,000 molecules/cell) in excess of IL-2R beta (11,000 molecules/cell). Accordingly, they display a number of beta/gamma complexes of intermediate affinity for IL-2 and IL-15 which is equivalent to the number of beta-chains. Both cytokines compete for binding to this beta/gamma complex. There are about 800 high affinity IL-15 receptors, suggesting the presence of a similar number of IL-15R alpha-chains. Within the common intermediate affinity beta/gamma-complex, the anti-beta-chain A41 mAb defines an epitope which is similarly engaged in IL-2 and IL-15 binding, whereas the anti-beta-chain 284 mAb defines an epitope which does not display similar interaction with either cytokines. Thus, although IL-2 and IL-15 compete for binding to this beta/gamma-complex, they do not use similar binding areas. Cross-linking and immunoprecipitation experiments have shown that the high affinity IL-15 receptors comprises IL-2R beta/gamma, in association with IL-15R alpha and that the three chains can be efficiently cross-linked to IL-15 and co-immunoprecipitated. Contrary to the intermediate affinity situation, high affinity IL-15 binding and subunit cross-linking were not affected by excess amounts of IL-2, A41 or 284 mAb, suggesting that when engaged in the IL-15 high affinity complex, the beta- and gamma-chains adopt different conformations, at least with respect to IL-15 binding. Finally, we provide evidence for the participation of a novel 35 kDa component within the high affinity structure. This component is immunoprecipitated with anti-IL-2R gamma mAb but not with anti-IL-2R beta mAb and might correspond to a truncated form of IL-2R gamma-chain.

Effect of cytokines and lipid mediators on the synthesis of interleukin 1 beta by human bone marrow stromal cells.

This study investigates the production of interleukin (IL-)1beta by cultured human bone marrow stromal cells. RT-PCR experiments indicate that two-thirds of cultures constitutively express IL-1beta mRNA transcripts. Their cell-associated IL-1beta levels are elevated after stimulation with tumour necrosis factor (TNF-)alpha but not with cytokines such as IL-1alpha, IL-3, IL-4, IL-6, IL-7, IL-10, SCF, G-CSF, M-CSF and TGF-beta or lipid mediators such as PGE2, LTB4, LXA4, LXB4, 12-HETE, 15-HETE and PAF. Addition of IL-4, but not IL-10 or TGF-beta, reduces the TNF-alpha-induced cell-associated IL-1beta. IL-1beta is never detected in bone marrow stromal cell supernatants whatever the stimulant added. In conclusion the pro-inflammatory molecule TNF-alpha stimulates bone marrow stromal cell-associated IL-1beta levels while the anti-inflammatory cytokine IL-4 reduces the TNF-alpha-induced effect. These results strengthen the key regulatory role of IL-4 on the production of haematopoietic cytokines by human bone marrow stromal cells.

Natural splicing of exon 2 of human interleukin-15 receptor alpha-chain mRNA results in a shortened form with a distinct pattern of expression.

We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15Ralpha) transcripts resulting from exon-splicing mechanisms within the IL-15Ralpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Ralpha bound IL-15 with high affinity, Delta2IL-15Ralpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ralpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15.IL-15Ralpha complex at the nuclear level. In sharp contrast, Delta2IL-15Ralpha was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Ralpha is a natural process that might play regulatory roles at different levels.

Mannose 6-Phosphate/Insulin-like growth factor II receptor mediates internalization and degradation of leukemia inhibitory factor but not signal transduction.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 subfamily of helical cytokines, all of which use the glycoprotein (gp) 130 subunit for signal transduction. The specific receptor for LIF, gp190, binds this cytokine with low affinity and is also required for signal transduction. We have recently reported that glycosylated LIF produced by transfected Chinese hamster ovary cells also binds to a lectin-like receptor, mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGFII-R) (Blanchard, F., Raher, S., Duplomb, L., Vusio, P., Pitard, V., Taupin, J. L., Moreau, J. F., Hoflack, B., Minvielle, S., Jacques, Y., and Godard, A. (1998) J. Biol. Chem. 273, 20886-20893). The present study shows that (i) mannose 6-phosphate-containing LIF is naturally produced by a number of normal and tumor cell lines; (ii) other cytokines in the interleukin-6 family do not bind to Man-6-P/IGFII-R; and (iii) another unrelated cytokine, macrophage-colony-stimulating factor, is also able to bind to Man-6-P/IGFII-R in a mannose 6-phosphate-sensitive manner. No functional effects or signal transductions mediated by this lectin-like receptor were observed in various biological assays after LIF binding, and mannose 6-phosphate-containing LIF was as active as non-glycosylated LIF. However, mannose 6-phosphate-sensitive LIF binding resulted in rapid internalization and degradation of the cytokine on numerous cell lines, which suggests that Man-6-P/IGFII-R plays an important role in regulating the amounts of LIF available in vivo.

Lipid mediators modulate the synthesis of interleukin 8 by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting interleukins (IL) such as IL-8. Lipid mediators modulate IL-8 synthesis in numerous cell types. We have investigated the effects of 5 lipid mediators (PAF, PGE(2), LTB(4), 12-HETE and 15-HETE) on the spontaneous and cytokine-induced IL-8 synthesis by human bone marrow stromal cells. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we demonstrate that these cells constitutively express IL-8 transcripts. By using a specific ELISA, we found that the production of IL-8 by marrow stromal cells is enhanced after stimulation with 12-HETE (1 microM) both in serum-free and serum-containing culture medium. LTB(4)(1 microM) enhances IL-8 production only in serum-supplemented medium. PAF, PGE(2)and 15-HETE (1 microM to 0.1 nM) have no effect on the spontaneous and serum-induced production of IL-8 by human bone marrow stromal cells. PGE(2)(1 microM or 10 nM) reduces marrow stromal cell IL-8 synthesis in response to IL-1alpha or TNF-alpha. In contrast, PAF, 12-HETE, 15-HETE and LTB(4)have no effect. In conclusion, various lipid mediators modulate the spontaneous, serum- or cytokine-induced IL-8 synthesis by bone marrow stromal cells, highlighting, for the first time, their potential role in the regulation of IL-8 production within the human bone marrow.

Binding of leukemia inhibitory factor (LIF) to mutants of its low affinity receptor, gp190, reveals a LIF binding site outside and interactions between the two cytokine binding domains.

The gp190 transmembrane protein, the low affinity receptor for the leukemia inhibitory factor (LIF), belongs to the hematopoietin family of receptors characterized by the cytokine binding domain (CBD). gp190 is one of the very few members of this family to contain two such domains. The membrane-proximal CBD (herein called D2) is separated from the membrane-distal one (called D1) by an immunoglobulin-like (Ig) domain and is followed by three fibronectin type III repeats. We used truncated gp190 mutants and a blocking anti-gp190 monoclonal antibody to study the role of these repeats in low affinity receptor function. Our results showed that the D1Ig region was involved in LIF binding, while D2 appeared to be crucial for the proper folding of D1, suggesting functionally important interactions between the two CBDs in the wild-type protein. In addition, a point mutation in the carboxyl terminus of the Ig region strongly impaired ligand binding. These findings suggest that at least two distinct sites, both located within the D1Ig region, are involved in LIF binding to gp190, and more generally, that ligand binding sites on these receptors may well be located outside the canonical CBDs.

Interleukin-4 (IL-4), but not IL-10, regulates the synthesis of IL-6, IL-8 and leukemia inhibitory factor by human bone marrow stromal cells.

Leukemia inhibitory factor (LIF), interleukin 6 (IL-6) and IL-8 are important regulators of inflammation and hematopoiesis. Human bone marrow stromal cells regulate marrow hematopoiesis by secreting cytokines. By using reverse-transcriptase polymerase chain reaction (RT-PCR), we demonstrate that human bone marrow stromal cells constitutively express LIF, IL-6 and IL-8 transcripts. By using specific ELISAs, we found that their spontaneous productions of LIF, IL-6 and IL-8 are elevated in response to serum and after stimulation with the pro-inflammatory cytokines IL-1alpha and TNF-alpha. The anti-inflammatory cytokine IL-4 reduces their serum- and cytokine-induced LIF secretion. By contrast, IL-4 stimulates their serum- and IL-1alpha-induced IL-6 synthesis. IL-4 has no effect on the serum-induced IL-8 synthesis by marrow stromal cells, but stimulates their cytokine-induced IL-8 production. The anti-inflammatory cytokine IL-10 has no effect on the serum- and cytokine-induced LIF, IL-6 and IL-8 synthesis by bone marrow stromal cells. RT-PCR experiments reveal the presence of IL-4 receptor alpha-chain mRNA and IL-10 receptor mRNA in cultured bone marrow stromal cells. The differential regulation by IL-4 of two related cytokines, such as LIF and IL-6, and the enhanced effect of this 'anti-inflammatory' cytokine on IL-6 and IL-8 synthesis highlight the tightly controlled regulation and the complexity of the cytokine production within the human bone marrow.

The mannose 6-phosphate/insulin-like growth factor II receptor is a nanomolar affinity receptor for glycosylated human leukemia inhibitory factor.

Comparison of the binding properties of non-glycosylated, glycosylated human leukemia inhibitory factor (LIF) and monoclonal antibodies (mAbs) directed at gp190/LIF-receptor beta subunit showed that most of the low affinity (nanomolar) receptors expressed by a variety of cell lines are not due to gp190. These receptors bind glycosylated LIF produced in Chinese hamster ovary cells (CHO LIF) (Kd = 6.9 nM) but not Escherichia coli-derived LIF or CHO LIF treated with endoglycosidase F. CHO LIF binding to these receptors is neither affected by anti-gp190 mAbs nor by anti-gp130 mAbs and is specifically inhibited by low concentrations of mannose 6-phosphate (Man-6-P) (IC50 = 40 microM), suggesting that they could be related to Man-6-P receptors. The identity of this LIF binding component with the Man-6-P/insulin-like growth factor-II receptor (Man-6-P/IGFII-R) was supported by several findings. (i) It has a molecular mass very similar to that of the Man-6-P/IGFII-R (270 kDa); (ii) the complex of LIF cross-linked to this receptor is immunoprecipitated by a polyclonal anti-Man-6-P/IGFII-R antibody; (iii) this antibody inhibits LIF and IGFII binding to the receptor with comparable efficiencies; (iv) soluble Man-6-P/IGFII-R purified from serum binds glycosylated LIF (Kd = 4.3 nM) but not E. coli LIF. The potential role of Man-6-P/IGFII-R in LIF processing and biological activity is discussed.

Increased levels of leukaemia inhibitory factor (LIF) in urine and tissue culture supernatant from human primary bone tumours.

Fifty-five adult patients with primary bone tumour, 27 benign and 28 malignant tumours, were assayed for leukaemia inhibitory factor (LIF) in urine and serum samples. Supernatant was obtained from primary tumour tissue cultures in 24 cases (14 benign, 10 malignant tumours). LIF was found in 11 urine samples (16.7%, 1 benign and 10 malignant tumours). In 23 urine samples from patients with malignant bone tumour tested before any treatment, LIF was detectable in eight cases (34.7%). High LIF levels were found in all supernatants from malignant tumour cultures and in supernatant from 12 of the 14 benign tumours cultured. LIF was never detected in control urine samples or supernatants from normal cancelous bone cultures. These first in vivo data concerning LIF in primary bone tumours raise the question as to the cellular origin of this multifunctional cytokine and its potential role in solid bone tumours and bone tumour resorption.

Epitope-function relationships of human leukemia inhibitory factor receptors using a novel set of anti-gp190 mAB.

The binding and functional properties of a set of six mAb directed against the human gp190 [leukemia inhibitory factor (LIF) receptor] signal transducing molecule were determined. Each of the antibodies reacted with a distinct epitope on gp190 expressed either by gp190-transfected Chinese hamster ovary cells or by the LIF receptor-positive choriocarcinoma JAR cell line. Two of the antibodies (1B4 and 6E6) had binding stoichiometries that were approximately 2-fold lower than those of other mAb (10B2, 12D9 and 7G7), suggesting either that gp190 is present as a pre-associated homodimer in the cell membrane or that part of gp190 is pre-associated with another component. Two mAb (1C7 and 1B4) were found to inhibit LIF binding on the two cell types studied. On JAR cells, this inhibition was, however, restricted to the high-affinity LIF component, suggesting different modes of LIF engagement with the low- and high-affinity receptor species. mAb 1C7 and 1B4 were also found to synergize for inhibiting LIF high-affinity binding. This synergy also extended to the inhibition of LIF- or oncostatin M (OSM)-induced proliferation of a Ba/F3 cell line co-transfected with human gp130 and gp190. However, this mAb combination inhibited LIF- but not OSM-induced haptoglobin secretion by HepG2 cells, suggesting that whereas haptoglobin secretion induced by LIF involves gp130/gp190 common LIF/OSM type I receptors, that induced by OSM mainly involves type II OSM receptors.

Leukaemia inhibitory factor (LIF) production in pleural effusions: comparison with production of IL-4, IL-8, IL-10 and macrophage-colony stimulating factor (M-CSF).

Leukaemia inhibitory factor (LIF), a pleiotropic cytokine detected in various inflammatory body fluids, plays a poorly defined role in the pathogenesis of human disease. This study was conducted to correlate the LIF concentrations in pleural effusions with the type of pathology and to compare its levels with those of IL-4, IL-8, IL-10 and M-CSF for a given pathology. Pleural fluids from 97 patients were assayed for cytokines by specific ELISAs. The concentrations of all cytokines tested were higher in infectious pleural effusions than in other pathologies (malignant or transudative). The lowest levels were observed for transudates. Significant differences were noted between pathology groups for each cytokine. A good correlation was observed between LIF and IL-8 for malignant effusions [regression correlation coefficient (RC) = 0.480, P < 0.01], between LIF and IL-4 for infectious disorders (RC = 0.543, P < 0.05) between LIF and IL-10 for transudates (RC = 0.798, P < 0.001) and between M-CSF and IL-8 in all pathologies tested except for primitive neoplasia (P < 0.05). The LIF concentration in pleural space seems to be strongly associated with the intensity of inflammatory reaction. The LIF production appears to have different regulatory patterns between aetiologic groups.

Leukemia inhibitory factor (LIF) and oncastsin M (OSM) high affinity binding require additional receptor subunits besides GP130 and GP190.

The structure of Leukaemia Inhibitory Factor (LIF) and Oncostatin M (OSM) receptors is not completely resolved. Heterodimerization of gp190 and gp130 has been proposed to form a high affinity receptor (type I) shared by LIF and OSM, while heterodimerization of gp130 with an as yet unidentified subunit is proposed to form a high affinity OSM receptor (type II) not shared by LIF. We have analysed the binding stoichiometries, cross-competition properties and cross-linking patterns of LIF and OSM to the choriocarcinoma JAR cell line. The data obtained are not fully accounted for by the model proposed above. They indicate rather that third chains of 140-150 kDa molecular mass, in addition to the gp130 and gp190 subunits, enter in the structure of LIF and OSM high affinity receptors. These results were strongly supported by transfection experiments in CHO cells. CHO cells co-transfected with the human gp190 and gp130 cDNAs expressed high affinity LIF receptors but no high-affinity OSM receptors, indicating that an additional component is required for high affinity OSM binding. High-affinity LIF cross-linking on these cells also showed the association of LIF with a 150 kDa component in addition to the gp130 and gp190 subunits.

[Modulation of integrin alpha-v-beta-1 expression on human tumor cells by leukemia inhibitory factor (LIF) and oncostatin M (OSM)]. / Modulation de l'expression de l'intégrine alpha-v-beta-1 à la surface des cellules cancéreuses humaines par le leukemia inhibitory factor (LIF) et l'oncostatine M (OSM).

Integrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell-cell or cell-extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be a requisite for the metastatic process. Treatment of the Foss human melanoma cell line with LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane alpha v beta 1 by 1.5-2 fold. The same phenomenon was observed on the SK-N-SH human neuroblastoma cell line. This modulation, which was inhibited by specific monoclonal antibodies against alpha v or beta 1 integrin subunits, was concomitant with improved tumor cell attachment to the fibronectin matrix. Similar results were obtained after TNF-alpha treatment. Our findings demonstrate the ability of LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.

Upmodulation of alpha v beta 1 integrin expression on human tumor cells by human interleukin for DA cells/leukemia inhibitory factor and oncostatin M: correlation with increased cell adhesion on fibronectin.

Integrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell-cell or cell-extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be requisite for the metastatic process. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane alpha v beta 1 1.5-2-fold. The same phenomenon was observed on the SK-N-SH human neuroblastoma cell line. alpha v beta 1 upmodulation was concomitant with improved tumor cells attachment to the fibronectin matrix. This greater adhesion of tumor cells to fibronectin was inhibited by specific monoclonal antibodies against alpha v or beta 1 integrin subunits. Similar results were obtained after TNF-alpha treatment. Our findings demonstrate the ability of HILDA/LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.

Modulation of LIF expression in human melanoma cells by oncostatin M.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are involved in stimulation of acute-phase protein during inflammatory processes. We recently suggested that these two cytokines may play a role in human immune surveillance during tumor progression. The present study was designed to determine the capacity of OSM in modulating LIF production by human tumor cells and to provide a better definition of the interactions between these two molecules during inflammatory reaction due to neoplasia. LIF content in culture supernatants was assayed by a specific ELISA (sensitivity: 25 pg/ml). In vitro exposure to recombinant OSM increased LIF production 2 to 6-fold in all human melanoma cell lines tested. This effect was not limited to melanoma cell types since LIF production was also enhanced by an MDA breast undifferentiated adenocarcinoma line. Moreover, a synergistic effect was observed for OSM and TNF-alpha. LIF increase was apparently due to up-modulation through LIF synthesis since LIF transcript expression was also enhanced in these tumor cell lines. It is concluded that OSM can induce inflammatory reaction not only directly but also via LIF production by tumor cells.

Human interleukin for DA cells/leukemia inhibitory factor and oncostatin M enhance membrane expression of intercellular adhesion molecule-1 on melanoma cells but not the shedding of its soluble form.

ICAM-1-mediated cell-cell adhesion is essential for different immunologic functions including non-MHC-restricted cytotoxicity. The shedding of a soluble form of ICAM-1 from melanoma impairs immune recognition and leads to tumour escape. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane ICAM-1 twofold without inducing sICAM-1 shedding. Conversely, TNF-alpha, in the same conditions, strongly stimulated membrane ICAM-1 expression and the shedding of the soluble form. The same phenomenon was observed on the A375 human melanoma cell line. ICAM-1 upregulation was concomitant with an increase in the non-MHC-restricted cytotoxicity of tumour cells mediated by LAK cell.s This higher sensitivity to LAK lysis was abolished by RR1/1, a specific monoclonal antibody against ICAM-1. Our results demonstrate for the first time the ability of HILDA/LIF and OSM to upregulate ICAM-1 expression on the melanoma cell surface, suggesting a potential role for these cytokines in human immune surveillance during tumour progression.

An ELISA for the measurement of human leukemia inhibitory factor in biological fluids and culture supernatants.

A new monoclonal antibody-based ELISA for leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) measurements is described. The sensitivity (56 pg/ml after 4 h incubation, 14 pg/ml after 24 h incubation), precision (intra-assays < 5%), reproducibility (interassay < 10%), and accuracy (recoveries, ranging between 98 and 119%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances guarantee accurate cytokine measurement in biological fluids such as serum, plasma, synovial fluid, follicular fluid, urine and culture supernatants. Using the assay, LIF/HILDA was measurable in supernatants after in vitro whole blood stimulation with phytohemagglutinin (PHA), OKT3, and phorbol myristate acetate (PMA) but not with lipopolysaccharide (LPS) or Ca ionophore. LIF/HILDA production was not measurable until after 24 h of culture, when cytokine levels were seen to increase linearly in the supernatant to reach values of up to 40 ng/ml after 96 h of culture. Finally, a good correlation was found (r = 0.96; p < 0.0001; y = 23.1x + 233) between the LIF/HILDA values obtained using the ELISA and DA-1a bioassay.

Generation of monoclonal antibodies against HILDA/LIF and their use in the quantitative assay of the cytokine.

An adoptive transfer immunization/fusion protocol in mice has been successfully used to raise a series of monoclonal antibodies directed against the Human Interleukin for DA-1a (HILDA)/Leukemia Inhibitory Factor (LIF) cytokine. These antibodies which were raised using recombinant HILDA/LIF purified from conditioned medium of transfected Chinese Hamster Ovary (CHO) cells also react with natural HILDA/LIF from the HSB2 T lymphoma cell line and unglycosylated HILDA/LIF produced in E. coli. They define four separate epitopes, one of which is involved in receptor binding and induction of biological activity. A sensitive sandwich immunoradiometric assay which is linear up to 5 ng/ml HILDA/LIF and can detect as low as 25 pg/ml of the cytokine has been developed.

High and low affinity receptors for human interleukin for DA cells/leukemia inhibitory factor on human cells. Molecular characterization and cellular distribution.

Radioiodinated recombinant human interleukin DA (HILDA)/leukemia inhibitory factor (LIF) purified from conditioned medium of Chinese hamster ovary transfected cells enabled the identification of specific receptor sites on a variety of human cell types. Using low concentrations (up to 500 pM) of the ligand iodinated at a high specific radioactivity, high affinity receptors (equilibrium dissociation constant Kd in the range of 30-100 pM) were first demonstrated. They were expressed at low levels by human peripheral blood monocytes but not by lymphocytes, NK cells, granulocytes, and platelets. The myelomonocytic cell line THP1 as well as the T lymphoma cell line HSB2 and the lymphoblastoid B cell line DAB were also receptor-negative. In contrast, most of the non-lymphoid tumoral cell lines tested, including melanomas, neuroblastomas, and carcinomas, expressed high affinity HILDA/LIF receptors at variable levels (Bmax from 20 to 600 sites/cell). The kinetics of HILDA/LIF high affinity binding to the choriocarcinoma JAR cell line were characterized at 4 degrees C with association and dissociation rate constants of k1 = 2.2 10(9) M-1 min-1 and k-1 = 0.0084 min-1, respectively, corresponding to a steady-state dissociation constant k1/k-1 = 3.8 pM. The subsequent use of higher concentrations of HILDA/LIF labeled at a lower specific radioactivity enabled the identification of a low affinity component on several cell lines (Kd in the range of 1-4 nM; Bmax from 1,000 to 5,000 sites/cell). On JAR cells, this low affinity component was characterized by association and dissociation rate constants at 4 degrees C of k1 = 7.3 10(7) M-1 min-1 and k-1 = 0.19 min-1, respectively (k-1/k1 = 2.6 nM). Affinity cross-linking of HILDA/LIF to JAR cells showed two cross-linked species under both reducing and nonreducing conditions corresponding to receptor species of 120 and 250 kDa, respectively. Whereas both bands had similar intensities under high affinity conditions, the higher band predominated under low affinity conditions. Our data suggest that the 250-kDa chain could constitute the low affinity binding component whereas the association of both 250- and 120-Da subunits would form the high affinity structure.

Macroporous calcium phosphate ceramic for long bone surgery in humans and dogs. Clinical and histological study.

Our previous studies reported the performance of Macroporous Biphasic Calcium Phosphate (MBCP) in spine fusion. In the present study, this material was used in block forms in selected patients with tumoral resection in long bone. Two cases were chosen with large benign bone tumors. Clinical and radiographic assessments, CT scans, and NMR were performed after 16 months, and in one case control biopsies were taken. In order to understand the kinetic process of biodegradation of the MBCP blocks and bone formation at the expense of the ceramics, an experimental study in surgically created bond defects in canine femoral cortices was made. The MBCP blocks recovered after implantation period from 2 to 18 weeks were analyzed using histological, stereological, ultrastructural, electron microprobe, and IR spectroscopy analyses. This study demonstrated the efficiency of MBCP blocks for filling pathological defects in human long bone. The biointegration process of the MBCP blocks was due to a partial dissolution of the ceramics crystals (b-TCP content) by multinucleated cells. Simultaneously, bone ingrowth at the expense of the ceramic is observed. The new bone formation inside the MBCP macropores and in the spaces between the blocks, involved the formation of a new cortical bone on the outer part, and a trabecularlike bone with bone marrow in the inner part of the implant. The biological resorption of the MBCP ceramic decreased after 1 month implantation in dog, due to the protective role of the newly formed lamellar bone on the surface and in the core of the ceramics.