Human Lymphokine Research Articles

GROWTH SUPPRESSIVE EFFICACY OF HUMAN LAK CELLS AGAINST HUMAN LUNG-CANCER IMPLANTED INTO SCID MICE

The purpose of our study was to determine the efficacy of immunotherapy using human lymphokine activated killer (LAK) cells against a human-lung squamous-cell carcinoma cell line (RERF-LC-AI) implanted into severe combined immunodeficient (SCID) mice. A statistically significant growth suppressive effect on RERF-LC-AI implanted into SCID mice was observed when human LAK cells were administered into the caudal vein of the mice treated with a continuous supply (initiated prior to LAK cells injection) of rIL-2. The human LAK cells stained with PKH 2, a fluorescent dye, for later detection using flow cytometry were administered into the caudal vein of RERF-LC-AI bearing SCID mice; the cells persisted for 7 days in the implanted lung cancer tissue and in the mouse peripheral blood, but for 5 days in the mouse spleen. The number of infiltrated human LAK cells in each tissue increased dose-dependently with the number of injected cells. The results indicate that the antitumor effect most likely occurred during the early implantation period of the human LAK cells. These results demonstrate the applicability of this model to the in vivo study of human lung cancer therapy.

Interleukin-13 stimulates interleukin-6 production by human keratinocytes: Similarity with interleukin-4

Interleukin-13 (IL-13) is a recently described human lymphokine which is produced by activated T-cells. Its effect on the production of IL-6 by normal keratinocytes and keratinocyte cell lines of human origin was studied and compared to that of IL-4. IL-13, similarly to IL-4, stimulated IL-6 expression by these cells in a dose- and time-dependent manner. Contamination with endotoxin was excluded by the use of polymyxin B and heat-inactivated cytokines. Further, we showed that IL-13, like IL-4, not only stimulated IL-6 production but also was able to induce overexpression of this cytokine in response to an inflammatory signal such as lipopolysaccharide (LPS). In a previous study, we demonstrated that IL-13, by inhibiting IL-6 and other cytokines produced by monocytes, exhibited an ‘anti-inflammatory profile’ comparable to that displayed by IL-4. In contrast, we show here that IL-13, by stimulating IL-6 production by keratinocytes, may favour the installation of an inflammatory process at a local level and, here again, it acted like IL-4. Therefore, according to the type of target cell, these two ‘TH2 type’ cytokines induce similar opposing effects on IL-6 production and are likely to be important cytokines in the regulation of inflammation at both systemic and local levels.

Coordinate regulation of multiple human lymphokine genes by Oct-1 and potentially novel 45 and 43 kDa polypeptides.

Abstract The hematologic response to inflammation is critically dependent on the release of a family of glycoproteins termed CSFs. Central to this process is the coordinated T lymphocyte production of three of these proteins, granulocyte macrophage-CSF (GM-CSF), IL-3, and IL-5. In the course of our studies of the transcriptional regulation of IL-3 expression we noted that the same or similar proteins that bound to the downstream-activating region of the human IL-3 gene were also captured using DNA probes derived from sequences present in the GM-CSF and IL-5 genes. In this study, DNA-protein cross-linking techniques and reporter gene analyses were used to define the nuclear proteins that mediate transcriptional enhancement of these genes at one cis-acting site. These studies establish that Oct-1, a member of the POU family of transcription factors, binds to regions of the GM-CSF, IL-3, and IL-5 promoters and contributes to basal T cell gene transcription. In addition, although the c-jun proto-oncogene product was detected in stimulated human T cells, two additional T cell-specific proteins (45 and 43 kDa) bind to the GM-CSF, IL-3, and IL-5 promoters and are functionally and immunologically distinct from c-jun or OAP40 (jun-D). These proteins contribute to transcriptional enhancement of the IL-3 and GM-CSF genes in stimulated T cells. Together, these data suggest that the coordinate regulation of several lymphokine genes is based on responses to shared proteins. These studies further our understanding of the networks of transcriptionally active proteins responsible for the host response to inflammatory stimuli.

Expression of interleukin-6 gene in human astrocyte cell lineages

Expression of the lymphokine genes in human astroglial cell lineages was studied. Primers for 7 different human lymphokines, from interleukin-1a (IL-1a) to interleukin-6 (IL-6), were used to analyze RNA transcripts in 5 cultured human astrocytoma cell lines and 9 brain specimens by polymerase chain reaction (PCR). Two out of 5 unstimulated astrocytomas, U138MG and U373MG, expressed IL-6 genes. After stimulation with IL1beta, all astrocytoma and one neuroblastoma cell lines expressed IL-6 genes, but other leukemic cell lines did not show RNA transcripts of IL-6. In addition to the cultured cells, we examined IL-6 gene expression within human malignant astrocytoma, peritumoural brain and autopsied normal brains. The result shows that tumour and cells of the surrounding reactive lesion express IL-6 genes, but it is not expressed in normal brains. Next, the concentration of IL-6 in the supernatant of cultured cells was measured quantitatively by sandwich immunoassay, ELISA. IL-6 activity was present, but low in all astrocytomas with the exception of A172. Furthermore, the concentration of IL-6 increased markedly with the stimulation of IL-1beta in both a time- and dose-dependent fashion. A172 could also produce IL-6 as was seen in gene expression. From these results, it is suspected that astroglial cell-derived IL-6 may participate in local immune reactions accompanying infection, degeneration and malignancies in the central nervous system.

Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses.

The discovery of new cytokines normally relies on a prior knowledge of at least one of their biological effects, which is used as a criterion either for the purification of the protein or for the isolation of the complementary DNA by expression cloning. However, the redundancy of cytokine activities complicates the discovery of novel cytokines in this way, and the pleiotropic nature of many cytokines means that the principal activities of a new cytokine may bear little relation to that used for its isolation. We have adopted an alternative approach which relies on differential screening of an organized subtracted cDNA library from activated peripheral blood mononuclear cells, using the inducibility of lymphokine messenger RNAs by anti-CD28 as a primary screening criterion. The ligation of the CD28 antigen on the T lymphocyte by a surface antigen, B7/BB-1, expressed on activated B lymphocytes and monocytes is a key step in the activation of T lymphocytes and the accumulation of lymphokine mRNAs. Here we report the discovery by molecular cloning of a new interleukin (interleukin-13 or IL-13) expressed in activated human T lymphocytes. Recombinant IL-13 protein inhibits inflammatory cytokine production induced by lipopolysaccharide in human peripheral blood monocytes. Moreover, it synergizes with IL-2 in regulating interferon-gamma synthesis in large granular lymphocytes. Recent mapping of the IL-13 gene shows that it is closely linked to the IL-4 gene on chromosome 5q 23-31 (ref. 4). Interleukin-13 may be critical in regulating inflammatory and immune responses.

Inhibition by fibrin coagulation of lung cancer cell destruction by human interleukin-2-activated killer cells.

We examined the effect of fibrin coagulation on tumor cytotoxicity mediated by human lymphokine (IL‐2)‐activated killer (LAK) cells. LAK cells were induced from peripheral blood mononuclear cells (MNC) by culture with recombinant IL‐2 for 4 or 5 days, and LAK cell‐mediated cytotoxicity against tumor cells was assessed by 51Cr release assay in the presence or absence of plasma from normal subjects and lung cancer patients. Plasma did not affect the phase of induction of LAK activity by IL‐2, but dose‐dependently inhibited the effector phase of LAK cell‐mediated cytotoxicity against Daudi cells. Similar inhibition of LAK cell‐mediated cytotoxicity was observed on pretreatment of Daudi cells and human lung cancer cell lines with human fibrinogen plus thrombin. A parallel relationship was found between the amount of fibrinogen in plasma of lung cancer patients and inhibition of LAK cytotoxicity. This inhibition was reduced by addition of anticoagulants (heparin or argatroban). These findings suggest that fibrin coagulation on tumor cells protects them from LAK cell‐mediated tumor cytotoxicity in malignant lesions and that a combination of an anticoagulant drug and IL‐2/LAK therapy may be effective for treatment of lung cancer patients.

The human lymphokine leukoregulin induces cell resistance to complement-mediated lysis.

Leukoregulin (LR) is a lymphokine secreted by human natural killer (NK) cells. Its effect on the susceptibility of K562 human erythroleukemic cells to lysis by antibody and complement was examined. As reported here, treatment of K562 cells with LR for 60 min at 37 degrees C confers on them resistance to complement damage. The LR-induced state of complement resistance is transient and the cells recover within 4-6 h unless a second dose of LR is added. The protective action of LR was observed using both conventional 51Cr-release and trypan blue inclusion assays. The protein synthesis inhibitors puromycin and cycloheximide and the protein kinase inhibitors tamoxifen, polymyxin B and W-7, could each block this action of LR. Fewer membrane attack complexes were found, following complement activation, on LR-treated than control cells. These results suggest that LR increases the capacity of K562 cells to down-regulate complement activation or repair the complement damage, possibly by inducing synthesis of defense proteins and/or activation of protective protein kinases.

Neoplastic and reactive human astrocytes express interleukin-8 gene

Expression of lymphokine genes in the human astroglial cell lineage was studied. Primers for 9 different human lymphokines, from IL-1 alpha to IL-8, were used to analyze RNA transcripts in 5 cultured human astrocytoma cell lines and fresh brain specimens by PCR. mRNA transcripts for IL-8 were detected in all neuroglial cells. In addition to the cultured cells, we examined IL-8 gene expression within human malignant astrocytoma, peritumoral brain and autopsied normal brains. The result shows that tumor and cells of the surrounding reactive lesion express IL-8 genes, but it is not expressed in normal brains. Next, the concentration of IL-8 in supernatants of cultured cells was measured quantitatively by a solid phase ELISA assay. IL-8 activity was produced constitutively in all astrocytomas and increased markedly upon stimulation with IL-1 beta or TNF alpha, in both a time- and dose-dependent fashion. From these results, it is suspected that astroglial cell-derived IL-8 may take part in neutrophil-mediated inflammation which accompanies infection, degeneration and malignancy in the brain.

Design and evaluation of a two‐stage, cyclic, recombinant fermentation process

A two-stage, cyclic fed-batch fermentation process to produce recombinant human lymphokine was designed. The organism used in the study was Escherichia coli K-12 containing a temperature-sensitive walkaway plasmid bearing an insert which codes for a human lymphokine. Transcription of the recombinant gene is controlled by a lambda repressor/pL promoter system. The lambda promoter is regulated by the temperature-sensitive product of the cl857 gene at 30 degrees C, but at 42 degrees C the promoter is derepressed. The first or growth, stage of the process was maintained at 28 degrees C and operated in the fed-batch mode. The vessel was fed at a rate which gives a constant specific growth rate using a media designed to maintain a constant optical density OD(600) of 50. After the volume in the first stage reached the maximum working volume of the vessel (12 L), a portion of the vessel contents was transferred to the second stage. The second, or induction/product formation, stage also operated in the fed-batch mode, was kept at 42 degrees C, and was fed with a media that is conducive to recombinant human lymphokine synthesis. An optical density of more than 100 was consistently achieved in the second stage. Thirty cycles were completed with a consistent yield of human lymphokine and cell density in each cycle. The process was used to produce 200 L of OD(600) 50 material from the first stage in 10 days. The volumetric productivity (g lymphokine/L. day) of the two-stage, cyclic fed-batch process is twice that of a single-stage, fed-batch fermentation process.

High and low affinity receptors for the human lymphokine HILDA/LIF: Binding properties and molecular structure: [formula omitted] and [formula omitted]Unité INSERM 211, Plateau Technique CHR, Quai Moncousu, 44035 NANTES Cedex 01

High and low affinity receptors for the human lymphokine HILDA/LIF: Binding properties and molecular structure: [formula omitted] and [formula omitted]Unité INSERM 211, Plateau Technique CHR, Quai Moncousu, 44035 NANTES Cedex 01

Modulation of a human immunosuppressive lymphokine by monosaccharides

Soluble suppressor factor (SSF) is a recently purified human lymphokine produced by peripheral blood lymphocytes (PBL) in serum-free medium as a likely consequence of an autologous mixed lymphocyte reaction. Immunoregulatory actions of SSF include suppression of: polyclonal B cell activation, proliferative responses of normal PBL, and natural killer (NK) and antibody-dependent cellular cytotoxicity. We examined the ability of the monosaccharides fucose (Fuc), galactose (Gal), glucose (Glc), and mannose (Man) to reverse SSF-mediated suppression of NK activity. Fuc and Gal can partially or completely reverse SSF-mediated suppression at four effector:target cell ratios. Man and Glc were unable to significantly reverse SSF-mediated suppression. Fuc or Gal was added to PBL at various times after addition of SSF. SSF-mediated suppression of NK cytotoxicity becomes irreversible with respect to these monosaccharides during the first 24 hr of PBL exposure to SSF. To explore the mechanism behind this block of SSF-mediated suppression, Fuc or Gal (50 m M) was cultured with PBL for 24 hr before addition of SSF, or with SSF for 24 hr before addition to PBL. Our experiments indicate that SSF is directly interacting with these monosaccharides, and may function by recognizing specific sugar moieties on the surface of effector cells.

A Cocktail of Three Monoclonal Antibodies Significantly Increases the Sensitivity of an Enzyme Immunoassay for Human Granulocyte-Macrophage Colony-Stimulating Factor

A sensitive and specific two-site ELISA was developed for human granulocyte-macrophage colony-stimulating factor (huGM-CSF) based on monoclonal antibodies (mAbs) which have been selected for high affinities and different epitope specificities. Using a cocktail of three mAbs, both for coating and, in their labeled form, for detection, a major increase in sensitivity was achieved (20-fold) compared to a two-site assay employing two different mAbs (one for coating and one for detection). The assay is as sensitive as the most sensitive biological assays. Recombinant mammalian cell expressed and natural huGM-CSF can be reliably detected down to 100 pg/ml (7 pmol/l). In contrast to conventional bioassays, the ELISA is highly specific for huGM-CSF and does not detect other human lymphokines. Results from quantification of recombinant and natural huGM-CSF in ELISA and bioassay correlate.

Lymphocyte chemoattractant factor induces CD4-dependent intracytoplasmic signaling in lymphocytes.

At present, a naturally occurring soluble ligand for CD4 has not been well described. There is much evidence to indicate that MHC class II molecules can bind to CD4; however, binding of intact class II molecules under physiologic conditions has been difficult to demonstrate. We investigated whether a previously reported human lymphokine, lymphocyte chemoattractant factor (LCF), which was described for its effects on human CD4+ lymphocytes and monocytes, could associate directly with CD4 and induce the generation of second messengers. In the present study, we demonstrate that CD4 affinity-purified natural and recombinant LCF induced a rise in intracellular calcium and increased inositol trisphosphate generation in normal human CD4+ lymphocytes and murine T cell hybridomas infected to express human CD4. Cell lines lacking human CD4 or expressing human CD4 molecules that lack the intracytoplasmic domain did not demonstrate a change in either calcium or inositol trisphosphate. The effect of LCF was blocked by coincubation with either anti-CD4 antibody or Fab fragments from anti-CD4 antibody. These studies demonstrate direct interaction of a lymphokine with CD4 and generation of second messengers as a result of the interaction.

Human recombinant interferon gamma enhances neonatal polymorphonuclear leukocyte activation and movement, and increases free intracellular calcium.

In previous studies, we have reported that after chemotactic factor stimulation, PMNs from neonates fail to undergo certain critical activation steps. Furthermore, the concentration of free intracellular calcium reached is significantly below that of PMNs from adults. Interferon-gamma (IFN-gamma) is a lymphokine that has been shown to activate phagocytic cells, and IFN-gamma messenger RNA production by neonatal mononuclear leukocytes has been reported to be depressed. In the present studies, we found that recombinant human IFN-gamma markedly enhanced the chemotactic responses of PMNs from neonates to levels that were not different from that of PMNs from adults. Furthermore, preincubation of the neonatal cells with this recombinant human lymphokine also corrected the abnormality in intracellular calcium metabolism. These results suggest that this developmental defect in phagocytic cell movement may be the result of an intrinsic defect in IFN-gamma production resulting in deficiency of this critical phagocyte-activating lymphokine.

Characterization of effector‐target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.

Human nonspecific suppressive lymphokines

Since the term "lymphokine" first appeared in print over 20 years ago, a tremendous number of these soluble mediators of the immune system have been described. Within the past few years, many human nonspecific suppressive lymphokines have been identified. This review discusses the historical basis of immunologic suppression and suppressor factors. Later reports describing suppressive human lymphokines are then grouped into four categories: primarily stimulatory lymphokines that also mediate certain suppressive activities, suppressive lymphokines produced during altered states of immunity, suppressive lymphokines produced by exogenously stimulated lymphocytes, and suppressive lymphokines produced by unstimulated lymphocytes. Recent work I have been involved in focusing on the human suppressive lymphokine soluble suppressor factor (SSF) is also discussed.

The enhancement of cytolytic activity in lymphokine activated killer cells using bispecific F(ab')2

In this study, we show that bispecific hetero-F(ab')2 enhances cytolytic activity in human lymphokine activated killer (LAK) cells derived from peripheral blood mononuclear cells against human small cell lung cancer (SCLC) cell lines. We used two types of bispecific F(ab')2 (anti-CD3-LU246mAb, anti-CD16-LU246mAb) which play different roles in the enhancement of cytolytic activity in LAK cells against the human SCLC cell lines N231, H69 and Lu135. Anti-CD3 Fab' or anti-CD16 Fab' were coupled with LU246 Fab' that recognized the antigen on human SCLC cells for reproducing bispecific F(ab')2. Anti-CD16 (3G8) Fab' conjugated with LU246 Fab' targeted NK-LAK cells to SCLC cells to mediate cytolysis, but NK-LAK cells induced by LGL-enriched fraction did not display enhanced cytotoxicity even when bispecific antibody was used in 51Cr release assay. Anti-CD3 (OKT3) Fab' conjugated with LU246 Fab' cross-linked T-LAK cells to SCLC cells activated T-LAK cells through the CD3 complex, and enhanced the cytolytic activity of T-LAK cells against SCLC lines. Although OKT3-LU246 F(ab')2 was not so potent in enhancing cytolytic activity in 51Cr release assay, it played a greater role in enhancing the inhibitory effect on tumor growth than 3G8-LU246 F(ab')2 in human tumor clonogenic assay and in vivo tumor neutralization assay. In addition, the enhancement of target cell lysis by bispecific antibodies was generally more potent than antibody dependent cellular cytotoxicity (ADCC) using LU246 monoclonal antibody.

IL-7 supports the generation of cytotoxic T lymphocytes from thymocytes. Multiple lymphokines required for proliferation and cytotoxicity.

Current data suggest that the combination of IL-2 plus IL-6 or IL-4 supports the generation of a CTL response. In this report, we investigated whether IL-7 alone or in combination with other lymphokines supports the generation of active CTL from murine thymocytes. Added alone, IL-7 produced a modest CTL response, and this response was augmented (more than an additive response) by the addition of either IL-2, IL-6, or IL-4. After culture with IL-7, the removal of CD8+ cells correlated with a marked decrease in killing, whereas removal of the CD4+ population resulted in an enhanced CTL response. IL-7 generated a proliferative response to which the addition of other lymphokines resulted in an additive increase at best. To determine if the CTL response was found with IL-7 was dependent on the presence of endogenous lymphokines, we added blocking mAb and found that anti-IL-2 or anti-IL-6 blocked the level of CTL generated in the presence of IL-7. In contrast, anti-IL-4 was unable to inhibit CTL generation. The effect of the mAb on proliferation was somewhat paradoxical. The addition of anti-IL-2, anti-IL-6, or anti-IL-4 to cultures containing IL-7 all caused a significant decrease in proliferation that was overcome by the addition of the corresponding human lymphokines for IL-2 and IL-6. If thymocytes were fractionated and then cultured in the presence of IL-7, the double negative population gave the greatest proliferative response, the single positive thymocytes gave a response that was approximately 40% less, and the double positive thymocytes did not proliferate. These data indicate that IL-7 supports the induction of a cytotoxic response, however, this appears to require the presence of both IL-2 and IL-6.

Human Recombinant Lymphokines and Cytokines Induce Pulmonary Eosinophilia in the Guinea Pig which Is Inhibited by Ketotifen and AH 21–132

Subcutaneous or intraperitoneal injection of recombinant human granulocyte-macrophage colony-stimulating factor, interleukin 3, or mouse tumour necrosis factor alpha, but not recombinant human interferon gamma, platelet-derived growth factor, or transforming growth factor beta caused selective eosinophilia of the pulmonary airways in the guinea pig. Unlike responses to platelet-activating factor, there was no attendant detectable airway hyperreactivity, but in common with responses to platelet-activating factor, eosinophilia of the airways was prevented by pretreatment with ketotifen or AH21-132. Cytokines or lymphokines may contribute to pulmonary eosinophilia in diseases such as asthma.

Chemostat study of kinetics of human lymphokine synthesis in recombinant Escherichia coli

Biotechnology and BioengineeringVolume 34, Issue 3 p. 415-421 Communications to the EditorFree Access Chemostat study of kinetics of human lymphokine synthesis in recombinant Escherichia coli C. E. Curless, C. E. Curless Amgen, Thousand Oaks, California 91320Search for more papers by this authorP. D. Forrer, P. D. Forrer Amgen, Thousand Oaks, California 91320Search for more papers by this authorM. B. Mann, M. B. Mann Amgen, Thousand Oaks, California 91320Search for more papers by this authorD. M. Fenton, D. M. Fenton Amgen, Thousand Oaks, California 91320Search for more papers by this authorL. B. Tsai, Corresponding Author L. B. Tsai Amgen, Thousand Oaks, California 91320Amgen, Thousand Oaks, California 91320Search for more papers by this author C. E. Curless, C. E. Curless Amgen, Thousand Oaks, California 91320Search for more papers by this authorP. D. Forrer, P. D. Forrer Amgen, Thousand Oaks, California 91320Search for more papers by this authorM. B. Mann, M. B. Mann Amgen, Thousand Oaks, California 91320Search for more papers by this authorD. M. Fenton, D. M. Fenton Amgen, Thousand Oaks, California 91320Search for more papers by this authorL. B. Tsai, Corresponding Author L. B. Tsai Amgen, Thousand Oaks, California 91320Amgen, Thousand Oaks, California 91320Search for more papers by this author First published: July 1989 https://doi.org/10.1002/bit.260340318Citations: 16AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume34, Issue3July 1989Pages 415-421 ReferencesRelatedInformation