Characterization of the human blood lymphocytes that produce a histamine-induced suppressor factor (HSF)

The cells which elaborate a soluble suppressor factor in vitro in response to histamine (histamine-induced suppressor factor or HSF) were partially characterized in the present studies. Human blood T- and B-cell populations were purified by affinity chromatography with rabbit anti-human F(Ab′)₂ and examined for their ability to make HSF. Highly purified populations of T cells, but not B cells, produced HSF in response to varying concentrations of histamine (10^?4 to 10^?4M). The HSF-producing cells were characterized further by means of affinity chromatography with columns containing conjugates of insolubilized histamine as well as by rosette formation with IgG (Tγ)- or IgM (Tμ)-coated ox red blood cells. These studies revealed the following: (a) Cells that synthesize HSF are retained on histamine (but not control) columns; (b) cells with histamine receptors comprise approximately 50% of the Tγ subpopulation but are not found in the Tμ subpopulation; (c) cells not retained by histamine columns have a reduced capacity to develop into suppressor cells following stimulation by concanavalin A or specific antigen (compared to unfractionated or control column passed cells). In addition, it was shown that cells synthesizing HSF predominantly express histamine type 2 receptors: (d)4-Methyl histamine (H₂ agonist), but not 2-methyl histamine (H₁ agonist), was capable of inducing HSF production; (e) cimetidine (H₂ antagonist) inhibited HSF production but chlorpheniramine (H₁ antagonist) did not. Taken together, these experiments suggest that T lymphocytes capable of expressing suppressor function following activation by histamine, specific antigen, concanavalin A, or perhaps through their Fc receptors may either be heterogeneous within the same subpopulation or more likely be the same cell with the complement of receptors described above.     

Partial characterization of a prostaglandin-induced suppressor factor

Glass adherent splenic T cells, cultured in the presence of prostaglandin E₂ (10^?5M), were found to elicit a factor capable of nonspecifically suppressing PHA- and LPS-induced mitogenesis. Cells from C57B1/6J, Balb/C, and C3H/He mice were all capable of producing this suppressor factor, although some degree of variability in the response of cells from C3H mice to the factor was observed. The suppressor (designated prostaglandin-induced T-cell derived suppressor, PITS) was characterized biochemically and it was found that the activity was resistant to boiling, and treatment with RNase and DNase, yet was sensitive to treatment with proteinase K, trypsin, and Pronase. Further, PITS supernatants were found to contain at least two suppressors with approximate molecular weights of 35,000 (PITS_α) and 5000 (PITS_β). Results from experiments with cycloheximide-treated glass-adherent T cells indicate that prostaglandin E₂ may function by inducing the release rather than de novo synthesis of the PITS. These results indicate that the reported overall suppressive effect of prostaglandin E₂ on lymphocytes may in part be due to the release by certain T cells of a suppressive factor.     

Properties of histamine-induced suppressor factor in the regulation of lymphocyte response to PHA in mice

Splenic T lymphocytes release a suppressor factor into the culture supernatant when incubated for 24 hr with histamine (10^?4M). Histamine-induced suppressor factor (HISF) inhibits lymphocyte response to PHA; it is released by T lymphocytes (either nylon-nonadherent or nylon-adherent lymphocytes) and not by B-cells or macrophages; its production is not observed after depletion of histamine receptor-bearing lymphocytes and is blocked by the H2 receptor antagonist (cimetidine) but not by the H1 receptor antagonist (diphenhydramine). Gel filtration by Sephadex G100 chromatography indicates that HISF had an approximate MW of 45,000 to 68,000. Its inhibitory activity was removed by passage over a histamine RSA-Sepharose column, but not by passage over rabbit anti-mouse Ig-Sepharose column; it was blocked by prostaglandin synthetase inhibitor (PGS) (Ro 205720) indicating that this activity is mediated by a prostaglandin (PG) synthesis.     

Augmentation of prostaglandin and thromboxane production in vitro by monocytes exposed to histamine-induced suppressor factor (HSF)

A possible mechanism to explain the suppression of mitogen-induced lymphocyte proliferation in vitro by histamine-stimulated mononuclear cells was investigated. In initial experiments, the inhibitory action of histamine-induced suppressor factor (HSF) on lymphocyte proliferation was documented to be reduced by the addition of indomethacin (1 μg/ml). Moreover, the addition of exogeneous PGE₂ (10^?7-10^?8 M) to mononuclear cell cultures reconstituted HSF activity in the presence of indomethacin. In order to ascertain the nature of the target cell responding to HSF, control and suppressor supernatants were incubated with human lymphocytes or monocytes (5 × 10⁶ cells/ml) for 24 hr. Following incubation, the supernatants were assayed for their content of prostaglandin E₂, F_2α, and thromboxane B₂. Monocytes (but not lymphocytes) incubated with supernatants containing HSF increased their production of prostaglandin E₂, F_2α, and thromboxane B₂ by 169, 53, and 49%, respectively. Suppressor supernatants were generated with histamine or an H-2 agonist (dimaprit) and chromatographed by gel filtration on Sephadex G-100. The elution profiles for the factor(s) inducing suppression of lymphocyte proliferation (25–40,000 daltons) and augmenting PGE₂ production (25,000 daltons) overlapped but were not identical. Collectively, these data suggest that HSF-mediated inhibition of lymphocyte proliferation may occur in part through the augmented production of prostaglandins and/or thromboxane B₂ by human monocytes.     

Partial purification and biochemical characterization of a T cell suppressor factor produced by human glioblastoma cells

The human glioblastoma cell line 308 constitutively secretes a soluble factor with biologic and biochemical characteristics of human monocyte-derived interleukin 1 (IL 1). The 308 cells also produce a 97,000 m.w. factor that inhibits the effects of IL 1 and interleukin 2 (IL 2) on T lymphocytes. By using sequential chromatography on Blue Affigel, hydroxyapatite, and Ultrogel AcA54, the inhibitory factor, termed glioblastoma-derived T cell suppressor factor (G-TsF), was separated from IL 1 and purified 2000-fold with respect to the protein present in the crude 308 cell supernatant. This G-TsF preparation was sensitive to tryptic proteolysis, showed a peak of pI 4.6 on isoelectric focusing, and when labeled with 125I, revealed six protein bands in the range of 30 to 100 kdaltons on SDS gel.     

Cellular interactions in the generation and expression of histamine-induced suppressor activity

The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymphnode were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT⁺ bone marrow cells, putative thymocyte progenitors, were MP⁺. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.     

SK&F 96365 inhibits histamine-induced formation of endothelium-derived relaxing factor in human endothelial cells.

Formation of endothelium-derived relaxing factor (EDRF) strictly correlates with the intracellular free Ca2+ ([Ca2+]i) concentration. We now demonstrate that the histamine-induced rise in [Ca2+]i of human umbilical vein endothelial cells is mostly due to activation of a membrane current which allows Ca2+ entry. This membrane current is sensitive to the novel inhibitor of agonist-induced Ca2+ entry, SK&F 96365, which blocked the histamine-induced sustained rise in [Ca2+]i, as well as 45Ca2+ uptake and membrane currents. Inhibition of the above cellular responses to histamine was accompanied by a considerable reduction of EDRF formation and release. Thus biosynthesis and release of EDRF from human umbilical vein endothelial cells significantly depend on agonist-induced Ca2+ entry involving receptor-operated Ca(2+)-permeable channels which can be blocked by SK&F 96365.     

Physiochemical characterization of lymphocyte inhibitory factor (LyIF) isolated from avian B and T cells

Thymic (T) or bursal (B) lymphocytes from chicks sensitized to Mycobacterium tuberculosis produce an avian lymphocyte inhibitory factor (LyIF). The physiochemical properties of both T and B LyIF were established by ultrafiltration which yielded four fractions with molecular weight ranges of greater than 100,000; 50,000-100,000; 10,000-50,000; and less than 10,000; enzymatic treatment with chymotrypsin and neuraminidase; varying pH; and heat exposure. These studies demonstrated that the maximum activity for both T and B LyIF was within a molecular weight range of 10,000-50,000. Both were sensitive to chymotrypsin and neuraminidase treatment. Both were stable at 56 degrees C for 30 min and resistant to changes in pH from 5 to 9. T-Cell migration was inhibited equally by B or T LyIF, while B-cell migration was inhibited to a lesser extent by T LyIF and B LyIF. Further experiments should establish the reasons for these observed differences in cross-reactivity.     

Preliminary characterization of human T cell suppressor factor (HTsF) isolated from tonsil cells by monoclonal antibody immunoadsorbence

We have shown that a murine monoclonal antibody, B16G, recognizes a constant region determinant of a T cell suppressor factor (TsF) in DBA/2 mice. The molecule recognized by B16G was shown to be a heterodimer with a native m.w. in the region of 80,000. We now show that B16G also reacts with a similar molecule derived from human lymphoid tissue. Yields of about 100 micrograms could be obtained from the solubilized membranes and cytosol from about 10(10) tonsillar cells by elution of adsorbed materials from B16G immunoadsorbent columns. As with the murine system, the human TsF (HTsF) thus derived was capable of suppressing the mixed leukocyte reaction (MLR) of homologous effector T lymphocytes. However, this same material was not suppressive across the HL-A barrier, that is, when allogeneic effector cells were used in the MLR. Preliminary characterization of the HTsF showed it to have a native m.w. of 80,000 to 90,000 to be composed of a heterodimer with subunit m.w. in the region of 45,000 to 50,000, and to have an associated peptide of approximately 25,000. These observations provide evidence for the conserved nature of genes encoding TsF and correlate with observations of other investigators on the considerable homology between genes encoding the murine and human T cell receptor.     

Regulation of human cytotoxic T-lymphocyte responses by a soluble suppressor factor

We describe some aspects of the biology of a suppressor factor (SF) secreted by actively metabolizing and dividing alloantigen-primed T cells which functions by regulating human cytotoxic T-lymphocyte (CTL) activation. The SF functions most effectively during the first 24 hr of CTL activation, while it does not function at the CTL effector stage. Both T cells and adherent cells are capable of absorbing out the biological activity from suppressor factor supernatants. Experiments demonstrated that either fresh adherent cells or the addition of interleukin 2 (IL-2) into the test system could reverse the effects of the SF on CTL activation. These data suggest that the SF could be acting by either indirectly restricting IL-2 availability to proliferating CTLs by limiting adherent cell interleukin 1 (IL-1) secretion or, alternatively, SF acting directly on the IL-2-producing T cells.     

Isolation and characterization of a T suppressor factor by using a monoclonal antibody

We have developed a monoclonal antibody to a T cell-derived suppressor factor (TsF) found in the serum of C57BL/6 mice hyperimmune to sheep red blood cells (SRBC). The antibody binds to the SRBC-specific TsF as well as to a TsF (TNP-TsF) from another system differing in both antigen specificity and MHC. It does not bind to unrelated proteins. The antibody inhibits the activity of the SRBC-specific TsF in vitro. By using the monoclonal anti-TsF, we can isolate sufficient quantities of TsF to demonstrate that it fulfills several properties that have been attributed to TsF, namely, MHC restriction, antigen specificity, and the requirement for a second chain. Also, the purified TsF gives a single 68,000 dalton band upon SDS-PAGE gel analysis under reducing conditions. We conclude, therefore, that we have a method of the isolation of pure TsF, as well as a probe for the genetic, biochemical, and biologic analysis of TsF.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-beta inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-beta receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-beta receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-beta whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-beta induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-beta acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-beta regulates angiogenesis. Finally, TGF-beta suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-beta pathway. In conclusion, TGF-beta signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.     

The isolation and characterization of the human suppressor inducer T cell subset

Immunization of mice with lower primate lymphoid cells has provided a useful strategy for raising monoclonal antibodies against functionally important surface determinants on human T lymphocytes. We have developed a monoclonal antibody, anti-2H4, which defines functionally unique human T cell subsets. This anti-2H4 antibody was reactive with approximately 42% of unfractionated T cells, 41% of T4+ inducer cells, and was reactive with approximately 54% of T8+ cytotoxic/suppressor population. Anti-2H4 was not reactive with human thymocytes, but reacted with subsets of peripheral blood B cells and null cells. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+2H4+ subset proliferate well to concanavalin A (Con A) stimulation, but poorly to soluble antigen stimulation, and provides poor help to B cells for PWM-induced Ig synthesis. The T4+2H4- subset, in contrast, proliferates poorly upon stimulation with Con A, but well on exposure to soluble antigen, and provides a good helper signal for PWM-induced Ig synthesis. What is, perhaps, most important, the T4+2H4+ subset functions as the inducer of the T8+ suppressor cells. Previous attempts to define the latter subset of cells has relied heavily on the use of specific autoantibodies present in the sera of patients with juvenile rheumatoid arthritis (JRA) and systemic lupus erythematosus (SLE). The present results suggest that anti-2H4 antibody defines the human suppressor induced subset of lymphocyte previously described as T4+JRA+. Last, the results reemphasize the previously documented remarkable structural conservation of certain T cell-specific determinants on lymphocytes of phylogenetically distant primates.     

Soluble suppressor factor produced by pokeweek mitogen-stimulated human peripheral blood leukocytes

Human peripheral blood leukocytes were exposed to either PWM or Con A mitogens. Cells activated by both these mitogens were able to depress proliferation in an MLC, and to inhibit the generation of spontaneous killer cell (SK) and induced T-cell cytotoxic activity. PWM-activated cells incubated in media for 48 hr were able to elaborate a soluble factor in vitro. This factor suppressed cytotoxicity, and was active only when present at the initiation of MLC cultures. In contrast, cells exposed to Con A were able to suppress immune responsiveness, but this population did not release a soluble factor which could inhibit cytotoxicity. PWM induction appears to be dependent on phagocytic cells, while Con A activation is less dependent on this adherent population. An enriched adherent cell population, stimulated with PWM, was able to suppress cytotoxicity. Thus, the PWM-stimulated system of suppression is mediated through a soluble factor and is dependent on adherent cells.     

Isolation and characterization of a tumor-specific T suppressor factor from a T cell hybridoma

In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.     

Refolding and structural characterization of the human p53 tumor suppressor protein

The human tumor suppressor p53 is a conformationally flexible and functionally complex protein that is only partially understood on a structural level. We expressed full-length p53 in the cytosol of Escherichia coli as inclusion bodies. To obtain active, recombinant p53, we varied renaturation conditions using DNA binding activity and oligomeric state as criteria for successful refolding. The optimized renaturation protocol allows the refolding of active, DNA binding p53 with correct quaternary structure and domain contact interfaces. The purified protein could be allosterically activated for DNA binding by addition of a C-terminally binding antibody. Analytical gelfiltration and chemical cross-linking confirmed the tetrameric quaternary structure and the spectroscopic analysis of renatured p53 by fluorescence and circular dichroism, suggested that native p53 is partially unstructured.     

Site-specific N-glycan characterization of human complement factor H

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.     

Purification and characterization of human urine-derived digitalis-like factor

We were able to purify a digitalis-like factor to apparent homogeneity from human urine based on the inhibitory effect on [3H]-ouabain binding to intact human erythrocytes. This ouabain displacing compound closely resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the Na+, K+-ATPase and serve as a specific regulator of the sodium pump.