Interleukin 2 produced by activated B lymphocytes acts as an autocrine proliferation-inducing lymphokine

Normal peripheral blood B cells produce a soluble factor after activation that is functionally indistinguishable from interleukin 2 (IL 2) and can support B cell proliferation in vitro. Purified rabbit peripheral blood B cells, when stimulated with a combination of ionomycin (0.5 microgram/mL) and phorbol myristate acetate (PMA) (1 ng/mL), secreted a soluble factor in the culture medium that supported the IL 2-dependent cell line CTLL-2. The ability of these supernatants to support CTLL-2 growth was almost completely blocked by rabbit antibodies against human recombinant IL 2 and by the anti-IL 2 receptor monoclonal antibody 7D4. These data strongly suggest that the growth factor secreted by rabbit B cells is IL 2. To examine the possibility that the IL 2 activity detected in the B-cell cultures may be derived from residual T cells, B cells were further purified by successive panning with a pan-T-cell monoclonal antibody, L11-135, and goat anti-rabbit IgG. These highly purified B cells produced levels of IL 2 activity comparable to those produced by the initial B cell populations. Comparison of IL 2 production by decreasing numbers of purified T cells and purified B cells also indicated that the B cells were the source of IL 2 activity. Supernatants of activated B cells could support proliferation of B-cell blasts, and this activity could be completely absorbed by CTLL-2 cells, indicating that IL 2 is a major growth factor for B cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human interleukin 1 is a cytocidal factor for several tumor cell lines

Highly purified interleukin 1 (IL 1) obtained from stimulated human monocytes appeared to be growth inhibitory and cytocidal for a human melanoma cell line, A375. Although IL 1 did not have an immediate cytolytic effect, with time in culture the growth of the target cells was irreversibly inhibited. The cells eventually lysed and decreased markedly in number; the IL 1 effect can therefore be said to be cytocidal. IL 1 activity could not be separated from the cytocidal activity by a variety of chromatography procedures by using conventional and high-performance liquid chromatography (HPLC). The A375 melanoma cell line was also sensitive to another human cytokine alpha-lymphotoxin (alpha-LT) derived from a human B cell line. IL 1 also appeared to be partially growth inhibitory and cytocidal for a LT-sensitive mouse fibroblast cell line, L929; but not for LT-resistant cells, including a subline of L929; a human epithelial carcinoma cell line, HeLa; a human osteosarcoma cell line, HOS; and a mouse SV40-transformed kidney cell line, TU5. However, the LT-sensitive mouse fibroblast cell line, L-M, was resistant to IL 1. Therefore, the cytocidal activity of IL 1 only partially overlapped the target cell selectivity of alpha-LT. Although natural IFN-alpha and recombinant IFN-beta were appreciably growth inhibitory for the A375 cell line, natural and recombinant IFN-alpha and recombinant IFN-beta and IFN-gamma exhibited little cytocidal activity. Purified IL 1 did not have any antiviral activity, and conversely, IFN and alpha-LT were not co-mitogenic for thymocytes. Furthermore, by ELISA and radioimmunoassays, antibodies against human alpha-LT, tumor necrosis factor, and IFN-gamma did not react with IL 1, indicating that IL 1 is antigenically distinct from these other cytokines. These in vitro results suggest that IL 1 may play a role in host defense against some tumors as a cytocidal factor.     

Stimulation of immunoglobulin secretion in human B lymphocytes as a direct effect of high concentrations of IL 2

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

Purification and characterization of a unique human interleukin 1 from the tumor cell line U937

Interleukin 1 (IL 1) produced by a human tumor cell line was purified to homogeneity by a three-step chromatographic method and was tested in various assays for multiple biologic properties. The purified IL 1 stimulated the proliferative response of the D10.G4.1 cell line, a mouse IL 1 indicator T cell; caused the release of prostaglandin E2 and prostacyclin from cultured human foreskin fibroblasts and from primary human umbilical vein endothelial cells; and elicited characteristic endogenous pyrogen fever in rabbits. To stimulate IL 1 production, the histiocytic lymphoma cell line U937 was incubated with the exotoxin from toxic shock strains of Staphylococcus aureus. Supernatants from stimulated U937 cells were concentrated, and were applied to a reverse-phase HPLC column. IL 1 activity was eluted from the column at high acetonitrile concentration. Subsequent chromatography over hydroxyapatite yielded a single IL 1 species with a pI of 5.5. IL 1 was then purified to homogeneity by gel exclusion HPLC migrating as a 14 kDa species. The molecular size was confirmed by SDS-PAGE and was visualized as a single molecule by silver staining; biologic activity was recovered from the same region of the gel. Limited N-terminal sequence analysis suggested some homology to the pI 7 form of the human blood monocyte IL 1. The pI 5.5 IL 1 produced by U937 cells was only partially neutralized with anti-human monocyte IL 1 antibody, suggesting that U937-derived IL 1 is structurally related to one of the molecularly cloned IL 1 species. IL 1 from stimulated U937 cells possesses the functional characteristics of monocyte IL 1 but may represent a structurally unique IL 1 species, as determined by sequence analysis, size, and antibody reactivity.     

Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

Purification of guinea pig tumor necrosis factor (TNF): comparison of its antiproliferative and differentiative activities for myeloid leukemic cell lines with those of recombinant human TNF

Recently, we suggested that the effect of differentiation inducing factor (D-factor) which is found in the supernatant of macrophages, and induced the differentiation of a mouse myeloid leukemic cell line, M1, into macrophage-like cells, may be a result of the cooperative effects of tumor necrosis factor (TNF) and interleukin 1 (IL-1). In this study, we purified guinea pig (G.P.) TNF secreted from peritoneal macrophages and compared the antiproliferative and differentiative effects of the G.P. TNF with those of recombinant human TNF (rHuTNF). The purification scheme consisted of ultrafiltration, gel filtration-high performance liquid chromatography (HPLC), DEAE-HPLC, and reverse-phase HPLC. The cytotoxic activity of the purified substance was approximately 1.5 x 10(8) U/mg. The isoelectric point was 5.2. The molecular weight was 40 to 45 kDa as estimated by gel filtration and 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence was determined to be Ser-Ala-Ser-Gln-Asn-Asp . . . . Approximately 76 or 71% homology between G.P. TNF and mouse or human TNF exists in the NH2-terminal 21 residues. The purified G.P. TNF and rHuTNF demonstrated D-factor activity only in the presence of recombinant human IL-1 alpha in M1 cells. We also determined the effect of TNF on two human myeloid leukemic cell lines (THP-1 and U937). The purified G.P. TNF and rHuTNF inhibited the growth of U937 cells, but did not induce their differentiation. In THP-1 cells, TNF slightly inhibited the growth and induced differentiation. In mouse cell lines G.P. TNF was more effective than rHuTNF for differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

Expression and characterization of human factor IX and factor IX-factor X chimeras in mouse C127 cells

Human blood clotting factor IX, and two chimeric molecules of factor IX, in which the first epidermal growth factor-like domain or both epidermal growth factor-like domains have been replaced by that of human factor X, have been expressed in mouse C127 cells. The recombinants have been purified using a metal ion-dependent monoclonal antibody specific for residues 1-42 of human factor IX. All recombinant molecules are activated normally by human factor XIa in the presence of calcium ion. Activation of the factor IX recombinants by factor VIIa-tissue factor appears to be normal for the epidermal growth factor-1 exchange but considerably reduced for the construction containing both epidermal growth factor-like domains of factor X. The analysis of gamma-carboxyglutamic acid residues reveals that all of the purified recombinants are almost fully carboxylated. The extent of aspartic acid hydroxylation at residue 64 is 60% for all recombinants. The chimeric molecule with both epidermal growth factor-like domains from factor X has about 4% normal activity in the activated partial thromboplastin time assay. In contrast, the construct containing the first epidermal growth factor-like domain of factor X shows essentially normal clotting activity. Thus, it is unlikely that this domain is involved in a unique interaction with factor VIII.     

Separable helper factors support B cell proliferation and maturation to Ig secretion

The 24-hr culture supernatant of Con A-activated spleen cells (SN) contains helper factors that enable maturation to high-rate polyclonal Ig secretion and enhance proliferation in cultures of mouse B cells activated with the F(ab')2 fragment of class-specific rabbit antimouse IgM antibody (anti-Ig). When interleukin 2 (IL 2), also called T cell growth factor, is removed from SN by absorption with an IL 2-dependent cell line at either 4 degrees C or 37 degrees C, all the helper activity for anti-Ig-activated B cells is also removed. Partial removal of IL 2 results in partial removal of helper activity for B cells. However, the IL 2-depleted SN appears to contain another helper factor, TRF, that enables anti-Ig-activated B cell cultures to mature to high-rate Ig secretion. This TRF activity is revealed by adding purified human IL 2 or an IL 2-containing supernatant of a cloned, lectin-activated T cell hybridoma line (FS6-14.13) to Il 2-depleted SN, which restores the polyclonal antibody response to anti-Ig. The hybridoma supernatant by itself supports proliferation of anti-Ig-activated B cell cultures, as measured by an increase in cell number, but not maturation to Ig secretion. This proliferative response is likewise IL 2 dependent, although purified IL 2 with anti-Ig is not sufficient. These experiments define separable combinations of factors acting on anti-Ig-activated B cell cultures, one of which (SN) results in both proliferation and maturation to high-rate Ig secretion, whereas the other (hybridoma supernatant) results in proliferation only. IL 2 appears to be an essential component of both combinations, although the target cell for IL 2 action in this system remains to be determined.     

Immunoreactive growth hormone production by human lymphocyte cell lines

1. Two human lymphocyte cell lines, a T-cell line and a B-cell line, were shown to produce and secrete immunoreactive growth hormone (irGH). The irGH molecules secreted by the two cell lines appeared to be de novo synthesized and their molecular size was similar to that of pituitary GH as well as irGH secreted by peripheral blood lymphocytes. 2. Affinity-purified irGH molecules had human growth hormone (hGH)-like mitogenic activity on Nb2 cells. These findings indicate that the irGH molecules produced by H9 and IM9 were similar to hGH in structure. 3. However, the irGH messages could not be amplified by polymerase chain reaction (PCR) primers which had been demonstrated to be able to amplify reverse-transcribed hGH messenger RNA successfully, suggesting that the lymphocyte-derived irGH and pituitary hGH are not exactly identical molecules. 4. We conclude that the H9 and IM9 cells produce a growth hormone-related molecule whose structure is different from that in the anterior pituitary.     

Interleukin 2 acts directly on a Tac-positive cloned B cell line established from a patient with adult T cell leukemia

A Tac-positive B cell line termed K3B was established from a patient with adult T cell leukemia (ATL). This cell line had EBNA antigen and human T cell leukemic virus (HTLV) provirus besides B1 antigen and surface immunoglobulin. A cloned Tac-positive B cell line termed K3B01 was obtained from K3B by the limiting dilution method. The K3B01 cells were shown to absorb IL 2 activity in a tonsillar IL 2 preparation. By using this cloned cell line and a purified recombinant IL 2 preparation, it was shown that the proliferation of K3B01 cells was enhanced by the addition of recombinant IL 2. Moreover, this response was inhibited by anti-Tac antibody. These results demonstrate definitively that IL 2 acts directly on B cells through IL 2 receptors on them.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

Production of a 26,000-dalton interleukin 1 inhibitor by human monocytes is regulated by granulocyte-macrophage colony-stimulating factor

An interleukin 1 (IL 1) inhibitor is secreted into culture medium by a human promyelocytic cell line, H-161, upon stimulation with (PMA) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Since the morphological characteristics of this cell line were macrophage-like, human monocytes were tested for their ability to produce similar activity using the same induction conditions. Upon induction of adherent peripheral blood monocytes with rhGM-CSF and/or PMA, an IL 1 antagonistic activity was found in the cell supernatants, as determined by IL 1 receptor binding assay, using the murine EL-4.6.1C10 cell line as the cell target. Most of the inhibition of IL 1 binding induced by PMA or by PMA/rhGM-CSF was shown to be caused by IL 1, since it was neutralized by a mixture of anti-IL 1 alpha/beta antibodies and was active in the murine thymocyte proliferation assay (LAF). The activity induced by GM-CSF alone was not neutralized by anti-IL 1 alpha/beta antibodies and showed no LAF activity. The IL 1 inhibitor activity was induced by rhGM-CSF with a D50 around 40 pg/ml. The activity was produced for more than 3 wk in the presence of GM-CSF; removal of GM-CSF was followed by a rapid decrease of IL 1 antagonistic activity. The specific binding of biosynthetically labeled IL 1 inhibitor to target cells (EL-4.6.1C10) showed a protein of 26 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This molecule shares biological and physical characteristics with the urinary IL 1 inhibitor and the promyelocytic H-161-derived IL 1 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources

Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP. The two GAP forms in placental extracts were resolved by molecular sieve chromatography and appeared to have a monomeric native structure. Human recombinant type I GAP was produced intracellularly in Sf9 insect cells using a baculovirus expression vector, and 10-mg quantities were purified to homogeneity in three steps. Comparison of the purified native and recombinant GAP molecules revealed that all three displayed similar biological specific activities in an in vitro GAP assay. A polyclonal antibody to purified recombinant GAP was prepared and shown to neutralize the activity of both native and recombinant GAPs. The antibody was also highly specific for the detection of native GAP by Western blot. Type I and II GAP species were detected in approximately equal amounts in cytoplasmic extracts of human placenta, but only type I GAP was observed when other human tissues were examined.     

A potential role for adrenocorticotropin in regulating human B lymphocyte functions

Adrenocorticotropin (ACTH) was found to enhance the growth and differentiation of human B lymphocytes. By using highly purified preparations of human tonsillar B cells, the effects of ACTH on the growth and differentiation of in vitro activated B cells were examined. Optimal concentrations of ACTH were found to increase the proliferation of activated B cells by twofold to threefold when ACTH was present in culture with either a B cell growth factor or recombinant interleukin 2 (IL 2). ACTH had essentially no effects when added to cultures of activated B cells in the absence of the growth factor. Additionally, when ACTH was added in conjunction with an optimal concentration of either a B cell differentiation factor or IL 2 to cultures of activated B cells, the combination of ACTH and factor enhanced Ig secretion by twofold compared with the factor alone. In the absence of the differentiative signal, ACTH had minimal effects on Ig production. Only the first 24 amino acid fragments of ACTH were required to enhance B cell growth and differentiation when combined with the appropriate, more classical signals. Thus, ACTH may have a physiologic role in regulating human B cell function.     

Evidence for shared antigenic determinants on rabbit and human interleukin 1 (IL 1)

An antiserum to human interleukin 1 (IL 1) was prepared by immunizing a goat with the isoelectric point (pI) 6.9 type of IL 1 in Freund's complete adjuvant. Serum-mediated inhibition of the biological activity of IL 1 appeared within 4 wk after the first immunization, and showed a progressive rise in titer over a 9-mo period. The inhibitory moiety was purified by sequential ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography, and the activity was found to co-purify with the IgG fraction of the serum. The antibody neutralized the biological activity of the pI 6.9 type of human IL 1 derived from either human placental tissue or human peripheral blood adherent cells, but did not neutralize the pI 5.2 type of IL 1 derived from either source. When used as an affinity reagent, the antibody selectively absorbed the pI 6.9 human IL 1, but not the pI 5.2 human IL 1. Furthermore, the antibody neutralized the pI 7.4 type of IL 1 derived from rabbit alveolar macrophages, but had no activity against the pI 4.6 IL 1 derived from the same source. No inhibitory activity against rat spleen cell-derived IL 1 or murine P388D1 cell line-derived IL 1 was detected. These experiments support the concept that the differing pI types of IL 1 derived from the same species are both biochemically and antigenically distinct molecules, and IL 1 of similar pI type derived from different species may share antigenic determinants.     

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.