Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range of 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes, and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

Disulfide assignments in recombinant mouse and human interleukin 4

The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined. The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays. Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients. Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction. These peptides were purified further and defined by composition and sequence analysis. Three sets of disulfide-linked peptides were consistently identified for each protein. For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined. The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines. A large double-loop region within the central three-fifths of each protein is stabilized by these bonds. Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells. Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 4

Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 92.2(1) A and c = 46.4(1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63% solvent, assuming there is one molecule in the asymmetric unit.     

Leukotriene B4 augments human natural cytotoxic cell activity

We have recently shown that leukotriene B4 (LTB4) activates T lymphocytes to become suppressor cells. We now report that LTB4 also augments human natural cytotoxic cell activity against target cells infected with herpes simplex virus. This activity is partially inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid and the thromboxane synthetase inhibitor OKY-1581, but is augmented by indomethacin. We suggest that LTB4 may play a role in early host defense responses during inflammatory and infectious disease processes.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Inhibitory activity of interleukin B on the suppressor T cell hybrid T2D4

Interleukin B (IL-B), a product of unstimulated B cells, is defined by its ability to selectively prevent the differentiation of suppressor T lymphocytes from precursors into effectors. The present study was undertaken to determine whether IL-B could also be active in modulating the activity of the T cell hybrid T2D4, which produces immunoglobulin-binding suppressor factors. T2D4 cells can be selectively induced by incubation with various isotypes of antibody to express isotype-specific Fc receptors and to release soluble factors that suppress production of the corresponding isotype. The data presented here demonstrate that IL-B is greatly effective in inhibiting T2D4 activities. Either pretreatment with IL-B or continuous exposure to IL-B prevents isotype activation of T2D4. As a result, T2D4 cells do not express isotype receptors and do not produce detectable amounts of isotype-specific suppressor factors. This IL-B regulatory activity on T2D4 is temperature dependent and is inhibited by cytochalasin B. These findings provide new insights on the mechanism by which IL-B enhances antibody responses, and they offer a conceptual framework for analyzing IL-B activity on suppressor T cells.     

Differential effects of recombinant human interferon-gamma and interleukin 2 on natural killer cell activity of peripheral blood in early human development

Ontogenic development and the lymphokine responsiveness of human NK cell activity against K562 target cells in peripheral blood lymphocytes were evaluated in fetuses, premature infants, and term neonates by using a 4-hr 51Cr-release assay. Basal NK activity and NK boosting by lymphokines were comparatively assayed after an 18-hr incubation with medium alone, recombinant human IFN-gamma (1000 U/ml), and recombinant human IL 2 (25 U/ml), respectively. Lymphocytes from 20-wk-old fetuses lacked NK cell activity even after the pretreatment with IFN-gamma. Low, but significant levels of NK activity and NK boosting by IFN-gamma were observed in premature infants after 27 wk of gestation, with a progressive intrauterine maturation of these activities. Both basal NK activity and NK boosting by IFN-gamma in term neonates were still lower than those of adult controls. The grade of NK boosting by IFN-gamma appeared to depend on the development of basal NK activity. Contrary to IFN-gamma, IL 2 could induce marked NK activity even in 20-wk-old fetuses who lacked both basal and IFN-gamma inducible NK activities. NK boosting by IL 2 was much more efficient than that by IFN-gamma at any period of human life. The facts that IL 2-induced NK boosting could occur without any appreciable production of IFN-gamma in neonatal lymphocytes, and that ample neutralizing doses of anti-IFN-gamma antibody hardly suppressed IL 2-mediated NK boosting even in adult lymphocytes, indicated that the effect of IL 2 on NK boosting might be independent of IFN-gamma production. On the basis of the ontogenic differences in the development of the lymphokine responsiveness of NK cell activity and on the different NK boosting mechanisms of these lymphokines it was suggested that so-called human "pre-NK cells" might be divided into IFN-gamma sensitive and IL 2-sensitive cells. Whether these cell populations belong to different cell lineages or different maturation stages of the same cell line, however, remains unsettled.     

Structure-activity relationships of recombinant human interleukin 2

Structure-activity relationships of recombinant human interleukin 2 were investigated by preparation, purification, and characterization of 21 missense mutants. A key role for residue Phe42 in the high-affinity interaction with receptor was indicated by (a) the reduction of 5-10-fold in binding affinity and bioactivity upon mutation of this residue to Ala and (b) the lack of evidence for conformational perturbation in Phe42----Ala in comparison with the wild-type protein as investigated by intrinsic fluorescence, second-derivative UV spectroscopy, electrophoresis, and reversed-phase HPLC, suggesting that the drop in binding is a direct effect of removal of the aromatic ring. In contrast, the conservative mutations Phe42----Tyr and Phe42----Trp did not cause significant reductions in bioactivity. UV and fluorescence spectra indicated approximately 60% overall exposure to solvent of tyrosines in the wild-type molecule, the tryptophan (residue 121) being buried; fluorescence data also showed that Trp42 in Phe42----Trp is likely to be within 1 nm of Trp121 and about 50% exposed to solvent. Phe44----Ala, Cys105----Ala, and Trp121----Tyr also exhibited reduced bioactivity, but these mutants are conformationally perturbed relative to wild type. None of the remaining mutants had detectably reduced bioactivity, even though several showed signs of altered conformation. Four mutants were recovered in very low yield, probably because of defective refolding.     

Keratinocyte growth-promoting activity from human placenta

Extracts of term human placenta were tested for enhancement of proliferative growth of primary cultures of human keratinocytes. Saline extracts or supernatants from homogenates were dialyzed extensively, lyophilized, and tested in subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only defined supplements (insulin, hydrocortisone, transferrin, ethanolamine, phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities (1000-3000 cells per cm2) formed colonies and grew in the presence of placental extract at 25-500 micrograms/ml. Extracts of cord serum or maternal serum were inactive, suggesting that the activity is derived from placental tissue. The activity is not EGF, since the activity in the placental extract, unlike EGF, did not promote growth at low cell density, was synergistic with EGF under some conditions, and did not produce changes in colonial morphology which occurred in the presence of EGF. Unlike keratinocyte growth-promoting activity in bovine hypothalamic extract, the activity is non-dialyzable and is destroyed at 100 degrees C. Placental extract could not replace any of the defined components of the medium and is therefore distinct from them. The presence of activity in the placenta with distinctive properties suggests that this is a previously undescribed material with growth-promoting properties for epithelium.     

Crystallographic characterization of recombinant human interleukin 2

Recombinant human interleukin 2 produced by Escherichia coli was purified to homogeneity and crystallized after being separated from methionyl interleukin 2. Crystals suitable for structural studies have been obtained by the seed enlargement technique, using the method of vapor diffusion with ammonium sulfate as the precipitant at pH 4.6. The space group is P2(1)2(1)2 with cell dimensions a = 49.2 A, b = 87.6 A and c = 32.4 A. The asymmetric unit contains one molecule of the protein. From preliminary results, the crystals are moderately stable to X-rays and produce measurable reflections to a resolution of about 2.2 A. The diffraction data for the native crystals have been collected on a diffractometer at 2.4 A resolution.     

The effect of recombinant interleukin 4 upon protein kinase activities associated with murine and human B lymphocyte plasma membranes

Exposure of plasma membranes isolated from high density resting murine B cells to recombinant IL-4 in the presence of gamma-[32P]-ATP promoted phosphorylation of a protein of Mr = 42,000. The 42 Kd protein kinase substrate could be detected in membranes prepared from low density B cells following a 24 h culture with lipopolysaccharide, but not in membranes prepared from B cells exposed to LPS for 48 h. Treatment of the cells with LPS resulted in the appearance of a number of new membrane-associated phosphoproteins. Treatment with the cytokine also resulted in the disappearance of a protein kinase substrate of Mr = 30,000 from phosphoprotein profiles of membranes prepared from cells exposed to LPS for 24 h. The 42 Kd structure appears to be a protein kinase substrate rather than possessing intrinsic phosphotransferase activity as judged from experiments employing 8-azido-gamma-[32P]-ATP as a photoaffinity label. No 42 Kd species was detectable using this reagent. Experiments employing identical protocols failed to reveal any enhanced or diminished phosphorylation of membrane-associated proteins in human peripheral blood B cells or in human B lymphoma cell lines.     

Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli

The lymphokine human interleukin 4 (IL-4) has been expressed from a plasmid in the cytoplasm of Escherichia coli. Advantage has been taken of insolubility of the human IL-4 in E. coli for rapid purification of this protein in only a few steps. We describe extraction and renaturation procedures which solubilize human IL-4 yielding biologically active protein. The protein was purified to homogeneity by one passage over a gel-filtration column. The refolded human IL-4 was characterized by N-terminal sequence analysis, amino acid analysis and bioassays. The refolded E. coli-derived human IL-4 has biological activity on T and B cells and binds to the human IL-4 receptor, comparable to mammalian expressed human IL-4, indicating that the protein is folded correctly.     

Recombinant interleukin 2 rapidly augments human natural killer cell activity

Recombinant human interleukin 2 (r-IL-2) rapidly stimulated human natural killer cell activity in vitro. Augmentation of NK activity occurred within 1 hr of preincubation with r-IL-2. Responsive killer cells were typical NK cells as shown by cell fractionation procedures. These included Percoll density gradient separation and depletion of OKT3+ T cells by an indirect rosetting method. Analysis with a panel of polyclonal and monoclonal antibodies against alpha and gamma interferon revealed that this early enhancement of NK activity by r-IL-2 was independent of the production of both types of interferon.     

High affinity binding of human interleukin 4 to cell lines

Purified human recombinant interleukin 4 (IL-4) was radio iodinated to high specific radioactivity without loss of biological activity. 125I-IL-4 bound specifically to the Burkitt lymphoma Jijoye cells and other cell lines. Jijoye cells showed a high affinity for 125I-IL-4 (Kd approximately equal to 7 10(-11) M) and displayed 1200-1400 specific receptors per cell at 4 degrees C or 37 degrees C. The equilibrium dissociation constant (Kd) corresponds to the IL-4 concentration which induces 50% maximal expression of the low affinity IgE receptor (Fc epsilon RL/CD23) on Jijoye cells. At 4 degrees C the rate constant of association K1 is 1.7 x 10(6) M-1 s-1 and the rate contant of dissociation k -1 is 1.3 x 10(-4) s-1 (t 1/2 = 91 min.) No human recombinant lymphokines other than IL-4 were able to compete for the binding of 125I-IL-4 to its receptor.     

The anti-tumour efficacy of human recombinant interleukin 2

Summary Experiments were designed to test what percentage of experimental MC-induced murine sarcomas were sensitive to the local tumour inhibitory effect of IL-2 and whether any correlation existed between the sensitivity of these sarcomas to the immunotherapeutic effect of IL-2 and their susceptibility to the cytolytic effect of IL-2-activated killer cells. It was found that the sensitivity of MC-induced sarcomas to local IL-2 immunotherapy was a general phenomenon. Repeated peri-tumoural injections of RIL-2 inhibited the growth of five (MC11, MC13, MC14, MC15, MC16) out of six sarcomas in syngeneic mice. The sixth murine sarcoma (MC12) was found to be resistant to the tumour inhibitory effect of IL-2. Similarly, five (MC11, MC13, MC14, MC15, MC16) out of six murine sarcoma cell lines were sensitive to the cytolytic effect of IL-2-activated syngeneic killer spleen cells when examined in vitro, whereas the sixth (MC12) sarcoma cell line was resistant. These results suggest that LAK cells represent the effector cell mechanism responsible for the anti-tumour efficacy of local IL-2 immunotherapy and that in vitro testing of sensitivity to the LAK cell-mediated cytolysis may be used to detect tumours responding to IL-2 immunotherapy in vivo.Abbreviations IL-2 interleukin 2 - RIL-2 human recombinant interleukin 2 - LAK lymphokine-activated killer - MC 3-methylcholanthrene - B10 C57BL/10ScSnPh - MSV murine sarcoma virus (Harvey) - MEM minimal essential medium     

Differential effects of interleukin 2 vs B cell growth factor on human B cells

The effects of recombinant interleukin 2 (IL-2) and high m.w. (HMW) B cell growth factor (BCGF) were examined on normal human peripheral blood B cells activated with Staphylococcus aureus Cowan I (SAC). When SAC-activated B cells were separated into Tac-antigen (Tac-Ag)+ and Tac-Ag- fractions by a cell sorter, recombinant IL-2 induced only the Tac-Ag+ cells to proliferate, whereas both Tac-Ag+ and Tac-Ag- cells responded to HMW-BCGF (m.w. 60,000). Alternatively, SAC-activated B cells were separated according to density into three fractions: low density (large) cells (82 +/- 15% Tac-Ag+), intermediate density (medium) cells (45 +/- 13% Tac-Ag+), and high density (small) cells (less than 5% Tac-Ag+). Recombinant IL-2 enhanced proliferation of low density cells the most, intermediate density cells less, and high density cells not at all. HMW-BCGF induced all three fractions to proliferate to approximately the same degree. Finally, the effects of IL-2 and BCGF on the DNA and RNA content of the various fractions of B cells was examined. RNA content was greater in IL-2-stimulated B cells than BCGF-stimulated B cells, whereas DNA content was the same in both cell populations. IL-2 and BCGF may preferentially interact with different subpopulations of B cells. The interaction of IL-2 or BCGF with normal activated B cells may induce both similar and different intracellular events.     

Production of interleukin 1 activity by normal human peripheral blood B lymphocytes

Interleukin 1 (IL 1) production by normal human B lymphocytes was investigated. Normal human peripheral blood B lymphocytes were purified by sequential separation with the use of Ficoll-Hypaque gradient centrifugation, sheep red blood cell rosette formation, Percoll gradients, and treatment with monoclonal antibodies (anti-Leu-M1, B73.1, and T101) and complement. Both purified large B lymphocytes (BL) and small B lymphocytes (BS) produced IL 1-like (thymocyte co-mitogenic and fibroblast mitogenic) activities in response to lipopolysaccharide. Maximal production of IL 1 activity by both BL and BS occurred at 48 hr. The m.w. of IL 1 activities from both BL and BS were about 20,000 with high pressure liquid chromatography, and the major isoelectric point of BL- and BS-derived IL 1 activity was 7.0. A rabbit anti-human monocyte IL 1 antiserum inhibited the activity of B cell-derived IL 1, suggesting antigenic similarities of monocyte- and B lymphocyte-derived IL 1 moieties. These data suggest that normal B lymphocyte-derived IL 1 activity is biochemically and immunologically similar to monocyte-derived IL 1.     

The effects of interleukin 1 on human B cell activation and proliferation

The precise role of B cell surface immunoglobulin (slg) in the activation of B cells is unclear at present. In particular, it is uncertain whether ligands interacting with the B cell slg suffice to induce proliferation, or simply induce a state of activation in which the B cell becomes responsive to growth factors made by accessory cells. We have examined the effects of two ligands, Staphylococcus aureus Cowan strain I (SAC) and antihuman mu chain (anti-mu), which interact with B cell slg on highly purified human peripheral blood and tonsillar B cells cultured at low cell concentrations. The effects on B cell proliferation of these ligands alone or in combination with highly purified interleukin 1 (IL 1) or a supernatant of a human T-T hybridoma containing a B cell growth factor (BCGF) were studied. SAC with its high cell wall content of protein A triggered maximal B cell proliferation which was not increased further by IL 1 or BCGF. High concentrations of soluble F(ab')2 fragments of goat anti-mu chain also induced significant B cell proliferation. Lower concentrations of anti-mu resulted in little or no B cell proliferation but activated the B cell to a state of responsiveness to both IL 1 and BCGF. IL 1 by itself had no effect on the proliferation of unstimulated B cells or on the proliferation of in vivo-activated B cells which responded to BCGF in vitro, but demonstrated clear synergy with low concentrations of anti-mu antibody. BCGF alone augmented the proliferation of unstimulated B cells, presumably by acting on B cells which had undergone some degree of activation in vivo. In addition, it showed marked synergy with anti-mu antibody, which resulted in proliferation similar in magnitude to that induced by SAC. This synergy was far greater than that seen between anti-mu antibody and IL 1, and the resulting proliferative response was only slightly increased by the presence of IL 1. We conclude that the importance of accessory cell factors for the initial rounds of B cell proliferation depends on the strength of the initial slg-mediated activation signal. When this is strong, the response is maximal and independent of accessory cells or accessory cell factors. When it is suboptimal, a moderate synergy is seen with IL 1 and a dramatic synergy with BCGF.     

Development of immunoassays for human interleukin 3 and interleukin 4, some of which discriminate between different recombinant DNA-derived molecules

Two panels of hybridomas were produced that secreted monoclonal antibodies (MAbs) against recombinant DNA-derived human interleukin 3 and interleukin 4 (rhIL-3 and rhIL-4). From each panel, sensitive immunoradiometric assays (IRMAs) were developed which were capable of detecting the recombinant molecule used as the immunogen but were unable to recognize natural or other recombinant forms of the same cytokine. Subsequent studies using the MAbs from each panel showed that a number of the MAbs appeared only to recognize that particular recombinant molecule used as immunogen, with little or no binding to other recombinant forms of the molecule. By using MAbs that were found to be unrestricted in their recognition for different recombinant forms of the cytokines, it was possible to develop an IRMA for IL-4 that was capable of detecting natural IL-4 as well as all the recombinant forms equally. An IRMA was also developed for IL-3 but was not of equivalent sensitivity in detecting the different recombinant forms of IL-3 used in the study. The recombinant DNA-derived cytokine molecules used to raise the two panels of MAbs contained amino acid substitutions relative to the natural sequences, and these findings indicate that caution should be exercised when using immunoassays to estimate natural sequence molecules if antibodies raised to modified rDNA-derived molecules are used.