Therapeutic potential of carbon monoxide in multiple sclerosis.

Carbon monoxide (CO) is produced during the catabolism of free haem, catalyzed by haem oxygenase (HO) enzymes, and its physiological roles include vasodilation, neurotransmission, inhibition of platelet aggregation and anti-proliferative effects on smooth muscle. In vivo preclinical studies have shown that exogenously administered quantities of CO may represent an effective treatment for conditions characterized by a dysregulated immune response. The carbon monoxide-releasing molecules (CORMs) represent a group of compounds capable of carrying and liberating controlled quantities of CO in the cellular systems. This review covers the physiological and anti-inflammatory properties of the HO/CO pathway in the central nervous system. It also discusses the effects of CORMs in preclinical models of inflammation. The accumulating data discussed herein support the possibility that CORMs may represent a novel class of drugs with disease-modifying properties in multiple sclerosis.

HCG hastens both the development of mammary carcinoma and the metastatization of HCG/LH and ERBB-2 receptor-positive cells in mice.

Breast cancer is more frequent in human nulliparae, whereas its incidence is reduced by early fullterm pregnancy. Rodent studies suggest that chorionic gonadotropin secretion during pregnancy affords protection by inducing breast structure differentiation. Opposite effects, however, have been observed in cancer prone transgenic mice overexpressing the ß subunit of chorionic gonadotropin or pituitary luteinic hormone (LH). Here we assessed the effect of administration of human chorionic gonadotropin (hCG) for 21 days (corresponding to the duration of a mouse pregnancy) in virgin female mice transgenic for the activated rat (r-) ERBB-2 oncogene (BALB-neuT). In these mice, the onset of atypical mammary duct hyperplasia and its progression towards multiple mammary carcinomas is accelerated by hCG. hCG enhances the in vitro proliferation and in vivo metastatization of tumor cells from a BALB-neuT mammary tumor expressing the hCG/LH as well as the ERBB-2 receptors. These findings suggest that hCG favours the growth and progression of hCG/LH and ERBB-2 receptor-positive breast tumors.

Interleukin 12, interferon gamma, and tumor necrosis factor alpha are the key cytokines of the generalized Shwartzman reaction.

The Shwartzman reaction is elicited by two injections of lipopolysaccharide (LPS) in mice. The priming LPS injection is given in the footpad, whereas the lethal LPS challenge is given intravenously 24 h later. The injection of interferon gamma (IFN-gamma) or interleukin 12 (IL-12) instead of the LPS priming injection induced the lethal reaction in mice further challenged with LPS. Antibodies against IFN-gamma when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS, IL-12, or IFN-gamma. Antibodies against IL-12, when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS or IL-12 but not with IFN-gamma. These results strongly suggest that LPS induces the release of IL-12, that IL-12 induces the production of IFN-gamma, and that IFN-gamma is the cytokine that primes macrophages and other cell types. Upon LPS challenge, the lethal Shwartzman reaction is induced by a massive production of inflammatory cytokines that act on the target sites already sensitized by IFN-gamma. If mixtures of TNF and IL-1 or mixtures of TNF and IFN-gamma are used to challenge mice previously primed with IFN-gamma or IL-12, mortality is induced. In the same conditions, the individual cytokines or a mixture of IL-1 and IFN-gamma do not replace the LPS challenge. When the mice are primed with LPS, the combination of TNF, IL-1, and IFN-gamma induced only a partial mortality incidence suggesting that the involvement of other LPS-induced factors.

Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors.

Two novel human beta-chemokines, Ck beta-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ck beta-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1alpha, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.

Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin.

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.

The interferon gamma (IFN-gamma) receptor: a paradigm for the multichain cytokine receptor.

With the purification and cloning of the interferon gamma (IFN-gamma) receptor chains the mechanism of IFN-gamma action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-gamma) receptor complex consists of two chains: IFN-gammaR1, the ligand-binding chain, and IFN-gammaR2, the accessory chain. Binding of IFN-gamma causes oligomerization of the two IFN-gamma receptor subunits, IFN-gammaR1 and IFN-gammaR2, which initiates the signal transduction events: activation of Jak1 and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-gammaR1 intracellular domain on Tyr440 followed by phosphorylation and activation of Stat1alpha, the latent transcriptional factor. With all these steps established, the IFN-gamma receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex.

BACKGROUND: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS: The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.

T-cell precursors in mice bearing the Ehrlich ascites tumors.

Mice bearing Ehrlich ascites tumors and challenged with sheep erythrocytes produced fewer plaque-forming cells than did normal mice. At the same time the immunosuppression developed, the number of T lymphocytes in the thymus and spleen were reduced significantly. In the spleen, the number of B lymphocytes remained constant during carcinogenesis, whereas that of the macrophages increased significantly, as compared to the controls. In this paper, we demonstrated that the mechanism responsible for thymus and spleen depletion of theta antigen-bearing cells had to be ascribed to fewer T-lymphocyte precursors in the bone marrow of mice with cancer. The reduction of T-lymphocyte precursors was probably caused by the same "soluble factor(s)" produced by Ehrlich ascites tumor cells, which also interfered with the proliferation of myelopoietic stem cells in the bone marrow of mice with this neoplasm, as we previously reported. By performing several reconstitution experiments of lethally X-irradiated hosts, we determined that the immunodepression by Ehrlich ascites tumor cells was readily reversible, and the alteration of the T:B lymphocyte ratio in the spleen had a minor function, if any, in the pathogenesis of the immunosuppression.

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver.

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.

Paradoxical antidiabetogenic effect of gamma-interferon in DP-BB rats.

Previous studies have shown that anti-gamma-interferon (IFN-gamma) antibody reduces the frequency of autoimmune IDDM in the DP-BB rat. We tested the effects of systemically administered recombinant rat IFN-gamma in both DP-BB and DR-BB rats. Unexpectedly, IFN-gamma markedly reduced the incidence of IDDM as compared with control rats when administered six times per week at a dosage of 280,000 U between ages 30-35 to 105 days or ages 60-64 to 105 days. A lower dosage (28,000 U on alternate days) was also protective when administered to DP-BB rats between birth and age 60 days. However, long-lasting protection against IDDM development over the 1-year study period was achieved only by the highest dosage of IFN-gamma administered from age 30 to 105 days. Ex vivo production of tumor necrosis factor-alpha from splenic lymphoid cells (SLCs) and peritoneal macrophages of the rats treated with IFN-gamma was comparable with that of controls; however, SLCs from the IFN-gamma-treated animals secreted lower amounts of IFN-gamma after stimulation with concanavalin A. IFN-gamma treatment also markedly reduced the frequency of phenotypically activated SLC-expressing class II antigens and interleukin-2 receptor. Finally, in agreement with the observed antidiabetogenic effects, exogenously administered IFN-gamma induced neither insulitis nor IDDM development in DR-BB rats, a subline of DP-BB rats in which autoimmune diabetes rarely occurs spontaneously but can be induced by administration of polyinosinic-polycytidilic acid.

GM-1, a clone of the monoblastic phagocyte U937 that expresses a large respiratory burst capacity upon activation with interferon-gamma.

The human cell line U937 was cloned and screened for the responsiveness to interferon-gamma (INF-gamma). The selected subclone, named GM-1, expressed a high density of IFN-gamma receptors and showed HLA typing similar to that of the parental line but was devoid of the Y chromosome. GM-1 cells display a promyeloid phenotype as revealed by flow cytometry using a panel of murine antibodies. Following treatment with IFN-gamma GM-1 cells differentiated to a more mature monocyte stage and acquired the capacity to mount a respiratory burst. After treatment with differentiation promotors, such as phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO), and retinoic acid, GM-1 showed a more limited respiratory burst capacity. Superoxide release in IFN-gamma-activated cells was stimulated with f-Met-Leu-Phe, C5a, or PMA. The development of the respiratory burst capacity was accompanied with the expression of cytochrome b558, a component of the phagocyte NADPH-oxidase. GM-1 cells are useful for the study of the effects of IFN-gamma on the respiratory burst. They are more sensitive and yield a more homogenous response to IFN-gamma than U937 cells. The phenotype of GM-1 cells was stable for more than 5 years.

Dendritic cells and MPIF-1: chemotactic activity and inhibition of endogenous chemokine production by IFN-gamma and CD40 ligation.

We have examined the biological activity of the CC chemokine myeloid progenitor inhibitory factor 1 (MPIF-1) on human dendritic cells. MPIF-1 has chemotactic activity on dendritic cells derived from either peripheral blood monocytes or cord blood CD34+ progenitors. However, chemokine treatment did not induce further cell activation or maturation. In addition, MPIF-1 is constitutively released by monocyte-derived dendritic cells but not macrophages or monocytes (resting or stimulated). The proinflammatory stimuli lipopolysaccharide and tumor necrosis factor alpha, which induced the release of monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and interleukin-8, did not affect MPIF-1 release. In contrast, CD40 ligation and interferon-gamma treatment, while stimulating the production of the other chemokines, caused a pronounced reduction of MPIF-1 transcript and protein release. Thus, in dendritic cells the regulation of the production and release of MPIF-1 is distinct in comparison to other CC and CXC chemokines.

High-affinity receptor for interferon-gamma (IFN-gamma), a ubiquitous protein occurring in different molecular forms on human cells: blood monocytes and eleven different cell lines have the same IFN-gamma receptor protein.

High-affinity receptors for human IFN-gamma were analyzed using 13 different cells, including blood monocytes. Scatchard analysis showed one high-affinity binding site for each cell. One cross-linked complex between IFN-gamma and the receptor was detected, although their apparent molecular masses were variable in different cells, as also confirmed in immunoblots of membrane extracts. Variations in molecular masses were abolished if N-linked glycosylation was absent. Stable tryptic fragments contained the intact binding site for IFN-gamma and antibody epitopes characteristic of the extracellular domain of the IFN-gamma receptor of Raji cells and were of different sizes only if glycosylated. In addition, Northern analysis showed the same mRNA encoding the high-affinity IFN-gamma receptor in each cell analyzed. Thus, all cells including blood monocytes express the same high-affinity IFN-gamma receptor protein. N-linked sugars may give structural stability to the IFN-gamma receptor and are unlikely to be directly involved in IFN-gamma binding.

Variable region cDNA sequences and characterization of murine anti-human interferon gamma receptor monoclonal antibodies that inhibit receptor binding by interferon gamma.

Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.

Different colony stimulating activity by tumoral ascitic fluid and conditioned media.

The effects of intraperitoneal Ehrlich ascites tumor (ET) growth on the kinetics of hemopoietic stem cells in the host bone marrow were studied using the spleen colony and the soft agar culture techniques. There is a decrease in spleen colony forming capacity of bone marrow of ET bearing mice, whereas in vitro assays of the committed macrophage granulocyte precursors, by the soft agar method, show that in the same circumstances a high yield of granulopoietic colonies can still be obtained. A shift of the CFU-c/CFU-s ratio from 15 to 36 thus occurs. Moreover, ascitic fluid from tumoral mice displays strong activity as CSF on normal mouse marrow, twice as strong as the standard mouse embryo CSF. When conditioned medium from cultures of ET cells (ET-CM) is tested, the pattern of agar colonies obtained is different from the previously obtained pattern of growth kinetics; furthermore many colonies are composed of undifferentiated cells. The hypothesis is suggested that among the variety of known CSF's, the ET-CM represents a unique factor, capable of inducing proliferation of marrow CFU-c, but only limited differentiation.

In vitro studies in nickel allergy: diagnostic value of a dual parameter analysis.

A comparison was made between the diagnostic value of assaying nickel-induced lymphocyte proliferation (lymphocyte transformation test, LTT) and migration inhibition factor (MIF) production in nickel contact sensitivity. Although lymphocyte proliferation was significantly increased in the group of patients with skin test reactivity to nickel, positive LTT were also frequently found in skin test-negative subjects: in 63% of subjects with and in 30% of subjects without a history of metal allergy. This would limit the value of the LTT as an in vitro correlate of skin test reactivity. However, in certain patients positive lymphocyte transformation may reveal nickel sensitization at a time of undetectable skin reactivity. Data obtained with the macrophage migration inhibition test (MMIT) showed a good correlation with nickel patch test reactions. Accurate determination of MIF became feasible by using cells from the human monocytoid cell line U937 as target cells in a microdroplet agarose assay. Using this MMIT, positive reactions occurred in 13% of the healthy controls and false-negative reactions were found in 26% of patients with positive skin test reactivity to nickel. As LTT and MMIT data appeared to be only weakly correlated in the individuals tested, a dual parameter analysis was performed. An excellent correlation [p = 1.8 (10(-8]] was found between skin test and in vitro reactivity for individuals with matching in vitro results (60% of all individuals tested). In those individuals with discordant in vitro data, skin testing will remain indispensable for diagnosing nickel allergy.

Prevention of spontaneous autoimmune diabetes in diabetes-prone BB rats by prophylactic treatment with antirat interferon-gamma antibody.

The role of endogenous interferon-gamma (IFN gamma) in the development of insulin-dependent diabetes mellitus (IDDM) in diabetes-prone BB rats was evaluated. Several groups of these animals were treated under different, experimental conditions with a purified polyclonal antibody (Ab), antirat IFN gamma. The results show that when administered at doses of 100 or 200 micrograms/week from the 30/33th until the 105th day of age, the anti-IFN gamma Ab reversibly reduced the incidence of IDDM compared to that in control rats treated with either irrelevant rabbit IgG or PBS. Moreover, when given up to the 105th day of age, these doses of anti-IFN gamma Abs exerted comparable preventive effects regardless of whether application started as early as within 24 h after birth or at the end of the prediabetic period (e.g. 70/75 days). In contrast, under none of the above experimental conditions did larger doses of anti-IFN gamma Ab (500 micrograms or 1 mg/week) exert antidiabetogenic effects in the BB rats. Apparently, this was due to the exuberant production of neutralizing Abs elicited by the large amount of the xenogeneic Ab injected. At histoimmunological analyses, the BB rats treated with 200 micrograms/ week anti-IFN gamma Abs from 30-80 days of age exhibited a milder insulitic process along with diminished spleen frequency of activated lymphoid cells (MHC class II and interleukin-2 receptor positive). Taken together, these results provide further in vivo evidence for the central pathogenic role of IFN gamma in BB rat IDDM and anticipate the usefulness of specific IFN gamma inhibitors in the prevention of the disease in the clinical setting. Defining novel and less immunogenic forms of specific IFN gamma inhibitors than xenogeneic Abs is important for improving the efficiency of anti-IFN gamma-oriented approaches.

The effects of a nonimmunogenic form of murine soluble interferon-gamma receptor on the development of autoimmune diabetes in the NOD mouse.

Previous studies have shown that in vivo treatment with antiinterferon-gamma (anti-IFNgamma) monoclonal antibodies (mAbs) prevents the development of autoimmune diabetes in NOD mice. Although these findings anticipate that specific anti-IFNgamma therapies may be useful for the prevention/treatment of human insulin-dependent diabetes mellitus, there are several reasons why the use of anti-IFNgamma mAb may be difficult in the clinical setting. With the aim to develop alternative forms of specific anti-IFNgamma therapies, we recently produced a nonimmunogenic form of the soluble IFNgamma receptor (sIFNgammaR) that binds and neutralizes murine IFNgamma with an affinity higher than that of anti-IFNgamma mAb. In this study we compared the efficacy of sIFNgammaR to that of two anti-IFNgamma mAbs (XMG 1.2 and AN-18) in the prevention of spontaneous and accelerated (cyclophosphamide-induced) forms of autoimmune diabetes in NOD mice. The results show that in the spontaneous model, sIFNgammaR could prevent histological and clinical signs of autoimmune diabetes as efficiently as the two mAbs. Under ex vivo conditions, sIFNgammaR exhibited a more powerful modulatory effect than XMG 1.2 mAb on cytokine secretion from splenic lymphoid cells, which resulted in a significant reduction of Concanavalin A-induced IL-2 secretion and an augmented release of both unstimulated and lipopolysaccharide-induced IL-6. Moreover, although both mAbs were immunogenic and elicited formation of high titers of anti-rat IgG, sIFNgammaR did not induce antibody formation. Unexpectedly, in the cyclophosphamide-induced model, sIFNgammaR turned out to be less effective than either of the two anti-IFNgamma mAbs. Taken together, these data support the role of IFNgamma in the pathogenesis of NOD mice, but, more importantly, suggest that a nonimmunogenic approach is possible to the diminution of the effects of IFNgamma in this model.

CKbeta8, a novel CC chemokine that predominantly acts on monocytes.

We have studied the biological properties of a new human CC chemokine, CKbeta8, consisting of 99 amino acids including six cysteines. CKbeta8 mRNA transcripts were induced in monocytes by IL-1beta and, to a lesser extent, by IFNgamma, and were detected in RNA extracted from normal human liver and gastrointestinal tract. CKbeta8 is chemotactic for monocytes, but is inactive on IL-2 conditioned T lymphocytes, eosinophils and neutrophils. Desensitization experiments indicate that CKbeta8 and MIP-1beta completely share receptors on monocytes and that the CKbeta8 receptor, which appears to differ from the known ones, is also recognized by MCP-1, MCP-2, MCP-3, MCP-4, MIP-1alpha and RANTES.

Interferon-gamma receptor extracellular domain-IgG fusion protein produced in Chinese hamster ovary cells as mixture of glycoforms.

Glycosylation of proteins fulfills important functions and because of its diversity contributes to apparent protein heterogeneity. We investigated the heterogeneity of a fusion protein comprising the extracellular domain of the human interferon-gamma (IFN-gamma) receptor and parts of the human IgG3 constant region, a potential IFN-gamma antagonist. The protein was produced in Chinese hamster ovary (CHO) cells and was secreted into the culture medium as an 175 kD glycoprotein. Glycosylation represented approximately one-third of the apparent molecular mass of the fusion protein, consisted of N- and O-linked carbohydrate moieties, and included sialic acid residues as part of both N- and O-linked oligosaccharides. Fusion protein forms with different apparent molecular masses and charges were separated by ion-exchange chromatography. Preparative electrofocusing revealed a wide spectrum of glycoforms. Glycosylation of the fusion protein and of soluble IFN-gamma receptors, comprising the extracellular domain of the native sequence, expressed in insect and CHO cells did not interfere with affinity of ligand binding.