Oncostatin M as a potent mitogen for AIDS-Kaposi's sarcoma-derived cells.

Oncostatin M, a cytokine produced by activated lymphoid cells, regulates the growth and differentiation of a number of tumor and normal cells. In contrast to its effects on normal endothelial and aortic smooth muscle cell cultures, Oncostatin M was a potent mitogen for cells derived from acquired immunodeficiency syndrome-related Kaposi's sarcoma (AIDS-KS). After exposure to Oncostatin M, AIDS-KS cells assumed a spindle morphology, had an increased ability to proliferate in soft agar, and secreted increased amounts of interleukin-6. Oncostatin M RNA and immunoreactive Oncostatin M protein were found in AIDS-KS-derived cell isolates. These results suggest that Oncostatin M may play a role in the pathogenesis of AIDS-KS.

Monoclonal antibodies to human major histocompatibility complex class II antigens.

The production and characterization of monoclonal antibodies to major histocompatibility complex (MHC) class II molecules have contributed significantly to delineating the number and structure of gene products of the D region of HLA. The "first generation" of class II monoclonal antibodies detected monomorphic determinants, often reacting with multiple class II loci gene products. Later generations of anti-class II monoclonal detected serologic specificities like those previously detected by human alloantisera. In addition, numerous antibodies have been generated against polymorphic HLA class II determinants which have not been previously described using human allosera. Biochemical analysis utilizing techniques such as radiolabeling and immunoprecipitation, one- and two-dimensional gel electrophoresis, Western blotting, limited N-terminal amino acid sequencing, and peptide mapping have localized the epitopes detected by polymorphic anti-class II monoclonals to various class II locus gene products. Such analyses with monoclonal antibodies have also identified class II gene products previously identified only with cellular reagents. The fact that several monoclonal antibodies with similar serologic specificity detect different class II locus gene products underscores the complexity of the HLA-D region and suggests some possible mechanisms for the generation of polymorphisms in this region.

Relationship of serum levels of oncostatin M to AIDS-related and classic Kaposi's sarcoma.

Serum levels of circulating oncostatin-M (OM) were compared among cases of Kaposi's sarcoma associated with acquired immune deficiency syndrome (AIDS-KS) and multiple controls, including a homosexual man infected with human immunodeficiency virus type 1 (HIV-1), an HIV-1-uninfected homosexual man, and a heterosexual man; and among classic KS cases and heterosexual controls. Cases were selected from abstracts collected by a population-based cancer registry and from local AIDS clinics. Controls for the AIDS-KS cases were matched to the cases by age, sex, and race and were either friends of the cases or residents from the cases' neighborhoods; controls for the classic KS cases were similarly matched, but were obtained solely from neighborhood residents. Blood samples were obtained from participants, serum levels of OM were determined by enzyme-linked immunosorbent assay (ELISA), and CD4 cell counts were obtained by flow cytometry. Geometric mean levels of OM were compared among the risk groups adjusted for age and CD4 cell count. No differences in adjusted OM levels were found between AIDS-KS cases and HIV-1-infected homosexual controls (8.4 pg/ml vs. 10.2) or between classic KS cases and controls (13.3 pg/ml vs. 9.6); however the HIV-1-infected controls (both homosexual and heterosexual) matched to the AIDS-KS cases had higher levels than did the HIV-1-infected cases and controls. Among the HIV-1-infected groups, an inverse correlation between OM and CD4 cell count was observed and was statistically significant for the cases. Among all heterosexual controls (matched to either case group), serum OM was inversely related to age.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of monoclonal antibodies reactive with human class II beta chains by two-dimensional electrophoresis and Western blotting.

We have used the Western blotting technique to examine B lymphoblastoid cell line (B-LCL) membrane proteins separated by two-dimensional gel electrophoresis specifically to analyze the binding patterns of monoclonal antibodies to separated HLA class II antigen beta (beta) subunits. The B-LCL LG-10 (homozygous for DR7), in which at least two sets of class II molecules can be distinguished on the basis of different electrophoretic mobilities, was examined with five monoclonal antibodies which detect monomorphic determinants. Four of the antibodies reacted with only DR beta subunits, while one antibody, XD5.A11, reacted with DR and with additional beta chains. Examination of two polymorphic monoclonal antibodies, SFR3-DR5, specific for HLA-DR5, and SFR3-PI.1, which reacts with a determinant absent from DR3 and DR7 homozygous lines, showed that both bind beta subunits from Swei, a DR5 homozygous line. Purification of a subpopulation of Swei class II molecules using an SFR3-PI.1 affinity column showed that the determinants recognized by SFR3-DR5, SFR3-PI.1, and a monomorphic monoclonal antibody reactive only with HLA-DR beta subunits of LG-10, reacted with identical beta subunits. Additional class II antigen subunits reactive with XD5.A11 were nonreactive with the polymorphic antibodies and the HLA-DR-specific monomorphic monoclonal antibody.

HLA-DQw3-related determinants: analysis of subunit and spatial relationships.

More polymorphism exists among DQ region gene products than is suggested by present serologic definitions of these class II molecules. DQ beta polymorphism among haplotypes carrying the DQw3 specificity is considerable. The TA10 specificity is present on one allele of at least three different DQ beta alleles that carry the DQw3 specificity. We have examined a series of monoclonal antibodies directed against different DQ beta alleles carrying the DQw3 specificity to determine subunit and spatial relationship among the epitopes detected by these antibodies. The antibodies were examined by Western blotting and for their ability to inhibit the binding of fluoresceinated antibodies on either TA10+ or TA10- DQw3 haplotypes. Our results reveal that (1) multiple DQw3-related epitopes exist; (2) several anti-DQw3-related antibodies generated against TA10- DQw3 molecules are unable to inhibit the binding of a TA10-specific antibody on a TA10+ haplotype while strongly inhibiting binding of an antibody detecting the reciprocal DQ beta polymorphism on a TA10- DQw3 haplotype; and (3) there is a strong requirement for three-dimensional conformation in the formation of the majority of the epitopes examined here. Analysis of previously published amino acid sequences for the haplotypes investigated here suggest that charge changes at amino acids 45, and 57, respectively, may have a significant effect in changing the spatial relationship between the DQw3-related epitope(s) and other polymorphic determinants on DQ beta chains.

Molecular complexity of HLA-DQw3: the TA10 determinant is located on a subset of DQw3 beta chains.

In attempts to examine the relationships between serologic and structural polymorphisms of HLA-DQ molecules we have analyzed several monoclonal antibodies generated against polymorphic determinants on HLA-DQ molecules. One antibody, SFR20-DQw3, has a serologic reactivity like that of the previously characterized anti-DQw3-like monoclonal antibody, IVD12, but differs from IVD12 in its affinity for DQw3 molecules associated with DR4 and DRw9 haplotypes. Two other monoclonal antibodies have identical serologic and molecular specificity, and react with a subset of DQw3 positive cells; they have been designated SFR20-DQ beta 5. Biochemical analysis of the DQ molecules carried by DQw3-positive cell lines associated with different DR haplotypes (DR4, DR5, DRw8, DRw9, DRw12), reveal the presence of at least three different kinds of beta chains carrying the DQw3 epitope. All the cell lines bound by SFR20-DQ beta 5 (DR5, DRw8, and DRw12) possess DQ beta chains of indistinguishable electrophoretic mobility, which are different from the DQ beta chains of DQw3 cell lines not bound by this antibody while DQw3 beta chains carried by DR4 and DRw9 haplotypes are distinct from DQ beta 5-positive BLCL and from each other. The serologic reactivity of antibody DQ beta 5 correlates perfectly with an RFLP of the DQ beta gene designated DQw3.1 (Kim et al.: PNAS 828139, 1985), and with the serologic specificity TA10 as defined during the Ninth International Workshop (Schreuder GMT et al.: Histocompatibility Testing 1984). SFR20-DQ beta 5 reacts with a separated beta chain by Western blot analysis. The finding of indistinguishable beta chain electrophoretic mobility for all DQ beta 5/TA10 positive cell lines tested provide the molecular basis for these specificities, and strongly suggest that antibody SFR26-DQ beta 5 detects a single allele of the multiple DQ beta alleles which can contribute to the formation of the DQw3 specificity.

Analysis of HLA-DQ molecules with a monoclonal antibody detecting a DQ polymorphism absent from DQW1 homozygous cells.

We have produced a monoclonal antibody detecting an HLA class II determinant absent from DR1,2,W6, and W10 (DQW1) homozygous B lymphoblastoid cell lines (BLCL). The antibody, SFR16-PI.2, immunoprecipitates molecules with electrophoretic mobilities of DQ (DS/DC) molecules from DR7 homozygous cell lines. SFR16-PI.2 binds more sets of class II molecules from internally labeled DR7 homozygous cell membranes than from externally labeled extracts from the same cell line. Depletion of DR molecules from internally-labeled membranes indicates that SFR16-PI.2, in addition to reacting with DQ molecules, also reacts with an epitope on a biosynthetic intermediate of DR molecules, which is lost on the mature DR molecule. SFR16-PI.2-reactive molecules were examined on a DR5 homozygous cell line, where they could be compared to those isolated by two other monoclonal antibodies with DQ specificity, namely IVD12 and Leu-10. All three monoclonal antibodies isolated two sets of DQ molecules with varying degrees of affinity for one of the molecules. The serological and biochemical data presented suggest that SFR16-PI.2 detects the alternate alleles of the locus encoding DQW1.

Detection of mycoplasma contamination lymphoblastoid cell lines by monoclonal antibodies.

We have produced two monoclonal antibodies, SFR1-Myco 1 and SFR1-Myco 2, that detect Mycoplasma fermentans found to contaminate lymphoblastoid cell lines (LCL). The specificity of these monoclonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoclonal antibodies with known reference strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycoplasma in these cells as determined by a standard mycoplasma detection assay. Because of the potential for widespread contamination of B lymphoblastoid cell lines transformed with Epstein-Barr virus-containing supernatants obtained from marmoset cell lines contaminated with M. fermentans, these monoclonal antibodies have value as screening reagents for this mycoplasma species in LCL.

HLA-DQ alpha polymorphisms: oligonucleotide probes characterize the contribution of first and second domains to electrophoretic variants.

Polymorphism is known to exist within the HLA-DQ alpha locus in the human major histocompatibility complex, although such polymorphism may be "silent" in standard HLA typing. However, DQ alpha polymorphism may be functionally significant, either through DQ alpha epitopes functioning directly in the immune response or by affecting tertiary conformation of Ia molecules through differential alpha/beta pairing. We have previously defined a particular DQ alpha polymorphism through reactivity with a monoclonal antibody and restriction fragment length polymorphism pattern. We now characterize this DQ alpha polymorphism through two-dimensional gel electrophoretic analysis and identify a subset of DQ alpha molecules with unique characteristics. Investigation of these allelic variants using synthetic oligonucleotide probe analysis of genomic DNA suggests a localization of the DNA region encoding the DQ alpha 5 epitope and suggests possible evolutionary mechanisms accounting for these unique patterns.

Nonrandom association of cellular antigens with HTLV-III virions.

Retroviruses are known to incorporate cellular antigens as they bud from infected cells. To identify the cellular antigens that associate with the AIDS-retrovirus, we evaluated a preparation of HTLV-III antigens with a panel of monoclonal antibodies reactive with a variety of antigens expressed on the H9 T-cell line used to produce the virus. Only monoclonal antibodies that identified HLA class-II antigens, beta-2 microglobulin, and a single anti-HLA class-I antibody were reactive in an ELISA of solubilized HTLV-III virus. No reactivity was seen with 11 monoclonal antibodies to T-cell antigens or with five antibodies to determinants on HLA class-I A or B molecules. These data suggest that on H9 cells the association of budding HTLV-III virions with cellular antigens may be a nonrandom process in which some HLA antigens, particularly class-II antigens, are selectively incorporated into the viral envelope. It is possible that a selective association of HLA class II antigens with budding HTLV-III virions may also occur for T cells infected in vivo, and could have relevance for the pathogenesis of this virus.

Monosomy 6 in a human lymphoma line induced by selection with a monoclonal antibody.

The human Epstein Barr Virus-superinfected B lymphoma cell line BJAB-B95.8.6 was mutagenized by gamma irradiation, and HLA mutants were selected with the HLA-Bw6-specific monoclonal antibody SFR8-B6. One of the mutants obtained, BM19, had lost one of the chromosomes 6 present in the wild type cells. Electrophoretic analysis of phosphoglucomutase isozyme PGM3 and erythrocyte glyoxalase 1 from both cells supports this conclusion. The HLA antigens expressed on BM19 were HLA-A2, B13, Bw4, C-, DR2 (questionable), DRw52 (weak) and DQw1. This constitutes one of the haplotypes of the wild type cells, the other (lost from BM19 cells) being HLA-A1, B35, Bw6, Cw4, DR5, DRw52 (strong) and DQw3. Possibilities to employ BM19 cells for the analysis of the major histocompatibility complex and other chromosome 6-encoded genes as well as their products are discussed.

Presence of brain-derived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay.

Brain-derived neurotrophic factor (BDNF) is one of several endogenous proteins that play key roles in neuronal development and homeostasis. We describe here the characterization and use of a sensitive and specific enzyme-linked immunoassay (EIA) for BDNF protein. Recombinant BDNF was detected at concentrations as low as 10 pg/ml, whereas the EIA did not detect NT-3, NT-4/5, or NGF at concentrations as high as 100 ng/ml. Because BDNF protein sequences are identical among humans, mice, and rats, we utilized the BDNF EIA to detect BDNF in the circulation or brain regions of these species. High concentrations of BDNF were detected in human and rat serum, and up to 50-fold lower BDNF levels were present in citrated human or rat plasma. The BDNF signal (66-141 pg/ml) in 20% human plasma was completely blocked by pre-exposure of plasma to a monoclonal antibody (Mab) specific for BDNF but not by exposure to 5-fold greater concentrations of an irrelevant Mab of the same isotype (IgG1). There was a significant and positive correlation (r = +0.86) between plasma levels of BDNF and serotonin, an indoleamine that is specifically released from activated platelets. These results are consistent with the view that the BDNF detected in human and rat plasma is derived from platelet degranulation, and that circulating levels of BDNF are negligible. In contrast to human or rat serum, mouse serum contained no detectable BDNF. However, BDNF protein was readily detectable at 108-256 ng/g of tissue in hippocampus, frontal cortex, and neostriatum of mice and rats. Thus, the failure to detect BDNF in murine serum was not due to an assay defect but highlights a significant species difference in the tissue-specific expression of BDNF that may be of biological importance. The presence of BDNF protein in blood and brain regions at quantities which greatly exceed those described for NGF confirm the abundant distribution of this broadly-acting neurotrophic factor.

Detection of oncostatin M in human plasma and serum by a sensitive enzyme immunoassay.

A sensitive and specific enzyme immunoassay was developed for detecting oncostatin M (OM) in human plasma and serum. The assay utilizes three anti-OM monoclonal antibodies that recognize mutually exclusive epitopes, including a neutralizing epitope. A sensitivity of 24 pg/ml was routinely obtainable. The assay showed no cross-reactivity with leukemia inhibitory factor (LIF) or interleukin-6 (IL-6), other members of the cytokine family that includes OM. The utility of the enzyme immunoassay (EIA) was demonstrated by detecting the time-dependent accumulation of OM in plasma from lipopolysaccharide (LPS)-treated human whole blood. The concentration of OM in human sera from normal donors was generally below the detection limits of the assay. However, concentrations of OM greater than 25 pg/ml were found in 17 of 212 serum samples from apparently normal donors. The detection of OM in human plasma and serum demonstrates that the EIA could be a useful tool in examining the role of OM in physiologic and pathologic states.

Abrogation of the antiproliferative activity of oncostatin M by a monoclonal antibody.

Oncostatin M (OM) is a novel cytokine which exhibits pleiotropic effects on a wide variety of normal and transformed cell lines. To determine some of the physiological functions of OM we have characterized several monoclonal antibodies to the recombinant molecule. Antibodies OM1 and OM2 bound native, but not denatured OM, suggesting they recognize non-contiguous epitopes. A third antibody, OM6, bound predominantly denatured OM. Of the two antibodies which detect discontinuous epitopes, OM2, but not OM1, was identified as a neutralizing antibody based on its ability to abrogate OM activity in the growth inhibition assay (GIA) and to inhibit OM binding in the radioreceptor assay (RRA). OM2 was equally effective in abrogating the functional effects of either natural or recombinant OM, thereby demonstrating that the active sites of these molecules are structurally similar, if not identical.

A monoclonal antibody directed against the HLA-Bw6 epitope.

A cytotoxic monoclonal antibody, SFR8-B6, produced by immunizing KGH rats with partially purified glycoprotein from JY, a human B lymphoblastoid cell line, detects HLA-Bw6, a unique public antigen on the HLA-B molecule. By cytotoxicity, SFR8-B6 has complete concordance with the presence of Bw6 as defined by alloantisera, and its reactivity segregates with HLA in families. Because sera detecting Bw4 and Bw6 are rare, this monoclonal antibody has value as a perpetual standard. In addition, SFR8-B6 offers potential as a probe for investigating the molecular relationships of HLA antigens.

Ia antigens in inbred rats and their relationship to the MLR phenotype.

Three different alloantisera were raised by using Ag-B/MLR disparate rats, and the cytotoxic activity remaining after absorption with erythrocytes to remove anti-Ag-B antibodies was examined. The alloantisera detected surface antigens present only on B cells and segregation studies demonstrated that the genes that code for these antigenic specificities were linked to the major histocompatibility complex. The reactivity of the alloantisera with splenic lymphocytes from a panel of strains representative of the currently known Ag-B groups showed that multiple specificities were present in two of the three antisera and that these specificities were shared by many inbred strains. The appropriate absorption studies showed, however, that each antiserum detected an unique specificity that was found only in those inbred strains that shared the same mixed lymphocyte reactivity (MLR) phenotype as the donor strain. The alloantiserum produced against the KGH strain inhibited the MLR reactions involving this strain only when it was used as the stimulating cell population. The antigens detected by the three alloantisera described here have the characteristics of Ia antigens, and they have tentatively been designated Ia.1 (ACI anti-KGH), Ia.3 (B3 anti-BN) and Ia.4 (MNR anti-DA).

The binding pattern of a neutralizing monoclonal antibody to mutant oncostatin M molecules is correlated with functional activity.

Oncostatin M (OM) is a member of the cytokine family that includes leukaemia inhibitory factor (LIF), granulocyte colony stimulating factor (G-CSF), and interleukin 6 (IL-6). We previously reported the characterization of a monoclonal antibody (MAb) to OM, termed OM2, which neutralizes its functional activity. To gain information about the epitope detected by this MAb, we utilized a sandwich enzyme-linked immunoassay (EIA) to examine OM2 binding on a series of mutant recombinant OM molecules generated by site-directed mutagenesis, encompassing amino acid insertions, deletions, or alterations throughout the molecule. Carboxy-terminal deletions of a putative amphiphilic alpha helix past residue 185 abrogated binding of the OM2 MAb; alteration of a hydrophobic residue in the helix to a neutral one also prevented antibody binding. Analysis of mutants in which cysteines involved in intrachain disulfide binding were changed to serines revealed that one of two disulphide bonds was essential for OM2 binding. Two mutant molecules containing deletions in the amino-terminal one-fourth of the molecule were not bound by OM2, while a third mutant OM molecule with a C-terminal proximal internal deletion outside of the amphiphilic alpha helix was bound. The binding pattern of MAb OM2 to the OM mutant molecules correlated well with their functional activities. The data suggest that residues in both the C-terminal alpha helix and N-terminal one-fourth of the molecule are involved in neutralizing antibody binding and functional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of oncostatin M secretion by human retrovirus-infected cells with potent growth stimulation of cultured spindle cells from AIDS-Kaposi's sarcoma.

Oncostatin M (OM), a 30-kDa glycoprotein, recently was identified as a major growth-promoting factor in the conditioned medium (CM) of the 38-0 cell line, a CD4,+ chronically human T lymphotropic virus type (HTLV)-II-infected, transformed T cell line. CM 38-0 induced the proliferation of spindle cells cultured in vitro from AIDS-associated Kaposi's sarcoma (AIDS-KS) cells. To determine how much of the AIDS-KS cell growth activity present in 38-0 CM was because of the presence of OM, we depleted OM by using specific mAb-affinity chromatography. OM purified from this CM stimulated AIDS-KS cell growth in a concentration-dependent fashion. The effluent, completely depleted of OM, failed to induce growth of AIDS-KS cells. To detect the constitutive release of OM by cells acutely or chronically infected with either HTLV-I, HTLV-II, or HIV-1, we utilized an enzyme-linked immunoassay. Whereas the chronically infected cells released significant levels of OM, the acutely infected cells released little or no OM. The presence of OM in HIV-1-infected T-cell CM correlated completely with AIDS-KS cell growth activity. Infrequently, low level AIDS-KS cell growth activity was seen in the absence of OM. This correlated with relatively high levels of IL-6 in the CM. In a CM-containing OM in the absence of detectable IL-6, a neutralizing antibody to OM completely abrogated KS cell growth activity. The presence of specific oncostatin M receptors on the KS cell lines was confirmed by cross-linking experiments. The results shown here suggest that T cells chronically infected with HIV-1 can secrete OM, which may play a role in the initiation or progression of AIDS-KS lesions, either alone, or in concert with IL-6.

Alternative splicing of HLA-DQB transcripts and secretion of HLA-DQ beta-chain proteins: allelic polymorphism in splicing and polyadenylylation sites.

HLA class II antigens are highly polymorphic cell-surface proteins involved in initiation and regulation of the immune response. Allelic sequence variation primarily affects the structure of the first external domains of alpha and beta component chains. Here we provide evidence for other types of allelic polymorphism for the genes encoding these chains. Sequences of two cDNA clones corresponding to HLA-DQB mRNAs from an HLA-homozygous cell line exhibit both alternative splicing and read-through of polyadenylylation. Furthermore, alternative splicing that deletes the transmembrane exon is associated with only a subset of HLA-DQB alleles, while the polyadenylylation-site read-through is found in a larger subset. This suggest that polymorphic cis-acting elements within the HLA-DQB gene control both processing steps. Proteins, presumably encoded by alternatively spliced mRNAs lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against HLA-DQ. These proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines that do not contain alternatively spliced mRNAs.