Enhancement of human NK and LAK cytotoxicity against HCMV-infected cells by rhamnogalacturonan: specificity of reaction.

Human peripheral blood mononuclear cells (PBMC) and their subpopulations obtained from healthy donors were used to study improvement of MHC-unrestricted cytotoxic reactions against cells infected with human cytomegalovirus (HCMV) at different multiplicities of infection. Natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity against HCMV-infected cells was greatly enhanced in the presence of rhamnogalacturonan (500 ng/ml). The increase of the multiplicity of infection from MOI 0.1 to 1.0 had only a slight effect on cytotoxicity enhancement by rhamnogalacturonan. The chemical specificity of interaction of rhamnogalacturonan with effector cells and virus-infected cells was found to be analogous to the interaction with tumor cells, i.e., both types of target cells must express a receptor for rhamnogalacturonan since rhamnogalacturonan-mediated enhancement of NK and LAK cytotoxicity against HCMV-infected cells was similarly inhibited by preincubation of CD56+ effector cells with 60% deacetylated D-mannose pentaacetate.

Anti-(transforming growth factor beta) antibodies with predefined specificity inhibit metastasis of highly tumorigenic human xenotransplants in nu/nu mice.

Monoclonal antibodies (mAb) were prepared against conjugated transforming growth factor beta 1 (TGF beta 1) peptides: amino acid positions 48-60 and positions 86-101. Two antibodies, mAb 16-3G1 [anti-(48-60)] and mAb 5-2G6 [anti-(86-101)] cross-reacted with native TGF beta 1, -beta 2 and -beta 3 (16-3G1) or only with native TGF beta 1 (5-2G6). Both mAb were used to characterize TGF beta-mediated effects on the metastatic potential in nude mice of human carcinoma cell line SLU-1 and its metastatic subline SLU-M1. Autocrine TGF beta 1-mediated up-regulation of cell proliferation and its suppression by anti-TGF beta antibodies in vitro was recorded for SLU-M1 cells whereas SLU-1 cell proliferation in vitro appeared to be refractory to anti-TGF beta antibodies and exogenous TGF-beta 1. However, the potential of s.c. tumours to develop distant metastases in nude mice was about the same for both cell lines. Development of primary tumours and distant metastases could be suppressed by treatment of mice with anti-TGF beta antibodies. Thus we assume that the metastatic potential of tumour cells is independent of TGF beta-mediated growth-regulation effects in vitro. The anti-TGF beta-induced suppression of tumour progression and metastasis in nude mice might rather result from stimulation of the immune surveillance. TGF beta-mediated autocrine down-regulation of MHC-unrestricted cytotoxicity of activated human monocytes and CD56+ LAK cells and its reversion by anti-TGF beta antibodies could be readily demonstrated. In all our experimental series, the neutralizing potential of both anti-TGF beta antibodies, though directed against opposite sites of the TGF beta 1 molecule, was very similar.

Human monoclonal antibody developed against a metastasizing paraganglioma: preparation and preclinical studies.

A tumor cell line, named HS, was established from a bone metastasis of a patient with metastasizing paraganglioma. In vitro immunization of normal human peripheral blood mononuclear cells by coculturing with viable HS cells, followed by fusion with mouse myeloma cells, yielded a stable human/murine heterohybridoma producing the highly specific monoclonal anti-body KM-155. This MAB KM-155 is a member of the IgG3 subclass and shows no alpha GAL glycosylation that is specific for mouse but not for human antibodies. In pilot preclinical studies it could be demonstrated that MAB KM-155 is highly efficient in targeting a KM-155 antigen-expressing human tumor developing in nu/nu mice after xenografting. Moreover, the growth of KM-155 antigen-expressing human tumors in nu/nu mice was largely inhibited when the concentration of circulating MAB KM-155 was maintained at a high enough level by serial injections.

IL-12-mediated activation of MHC-unrestricted cytotoxicity of human PBMC subpopulations: synergic action of a plant rhamnogalacturonan.

IL-12 mediated activation of human MHC-unrestricted cytotoxicity was studied with freshly isolated, highly enriched CD56 +CD3- NK cells (95.98%), monocytes/macrophages (90-95%) and CD3 + T cells (95-98%). Activation of NK cell cytotoxicity and monocyte cytotoxicity by IL-12 were independent of exogenous IL-2 and IFN gamma. Activation of CD3+T cells to MHC-unrestricted cytotoxicity required coactivation by anti-CD3 antibody. The enhanced cytotoxicities were directed against NK-sensitive as well as NK-resistant target cells and coincided with enhancement of effector cell/target cell conjugate formation. The specific cytotoxicity of all three activated effector cell populations was further increased in the presence of rhamnogalacturonan. These increases were based on an additional increase of effector cell/target cell conjugate formation that is based on rhamnogalacturonan-mediated bridging between effector cells and target cells. Simultaneous enhancement of cytotoxicity indicates involvement of receptors on effector cells cross-reacting with acetylrhamnose (6-deoxymannose) that might play an important role in human MHC-unrestricted cytotoxicity against tumor cells.

Comparison of hexose acetate-specific receptors isolated from human leukocytes showing competitive inhibition of human CD56+NK and LAK cytotoxicity.

Specific cytotoxicity of human CD56+NK and LAK cells was quantitatively inhibited by acetylated mannose, galactose and glucose (Scand. J. Immunol., in press). The respective NK cell receptors were isolated from human leukocyte lysates by affinity chromatography based on 60% deacetylated penta-acetates of mannose, galactose and glucose. All three affinity isolates contained a main component with +/- 87 kDa molecular mass exhibiting about the same patterns of isoforms at pI 4.90, 4.75, 4.60 and 4.50 in isoelectric focusing. Moreover, preincubation of tumor target cells with the three 87-kDa receptors revealed very similar inhibitory potentials for human NK and LAK cytotoxicity showing dose-dependent inhibition between 20 (no inhibition) and 700 pmol/ml (100% inhibition) receptor concentration. The data support the assumption that the three affinity isolates contain the same type of receptor directed against a unique epitope common to acetylated mannose, galactose and glucose.

Optimizing production of human monoclonal IgG antibodies by in vitro-primed human PBMC: influence of CD56+ NK cell depletion.

Freshly isolated human peripheral blood mononuclear cells (PBMC) were immunomagnetically depleted of CD56+ cells. When these CD56- PBMC populations were cultured in the presence of autologous donor serum, polyclonal activation with IL-2 and pokeweed mitogen (PWM) generally resulted in exclusive production of IgG antibodies. Fusion with SP2/O-Ag14 mouse myeloma cells was highly efficient and yielded a great number of IgG-producing heterohybridomas. These conditions were used for in vitro immunization with viable human HT29 tumor cells. After fusion, an increase in hybridoma clones producing IgG monoclonal antibodies (MAb) with HT29 specificity showing a higher portion of MAb binding to the surface of viable HT29 cells was recorded. This immunizing efficiency was not observed with HT29 membrane protein fractions or HT29 proteins integrated into ISCOM particles. Investigations with human anti-alpha Gal antibodies showed that the IgG antibodies produced by the human/mouse heterohybridomas did not contain the mouse-specific Gal alpha 1-3Gal epitope.

Chemospecificity and cross-reactivity of target cell recognition by human CD56+ NK and LAK cells.

Inhibition of specific cytotoxicity of highly purified (> 95%) human CD56+ NK and LAK cells against K562 tumour cells was studied with various sugar acetates. Maximum inhibitory specificity was obtained with 60%-deacetylated penta-acetates of mannose, galactose, glucose, or 80%-deacetylated penta-O-acetate of N-acetyl neuraminic acid. The inhibition was strictly dosedependent and 100% inhibition was achieved in the concentration range of 500-1000 nmoles/ml with all four sugar acetate samples. Enhancement of specific cytotoxicity in the presence of rhamnogalacturonan (RG; 500 ng/ml), acting as a bridging molecule, was also inhibited in a dose-dependent manner with the same inhibitory specificity and within the same concentration range indicating involvement of the same number of sugar acetate-specific receptors. Moreover, formation of lytic CD56+ effector cell/tumour cell (E/T) conjugates was equally well inhibited whereas formation of total E/T conjugates was only partially inhibited (NK: 44-73%; LAK: 46-50%). E/T conjugate formation in the presence of RG was enhanced. Inhibition of the enhancement of formation of lytic E/T conjugates in the presence of RG was again completely accomplished with the same inhibitory specificity and within the same concentration ranges as recorded for E/T conjugate formation in the absence of RG. However, inhibition of total E/T conjugate formation was again only partially achieved at the given concentrations. The data support the assumption of an NK cell receptor with specificity for acetylated carbohydrate moieties on target cells or on bridging molecules such as RG.

Structural specificity of MHC-unrestricted recognition of HCMV-infected target cells by human CD56+NK and LAK cells.

Structural specificity of binding and cytolysis of HCMV-infected human foreskin fibroblasts (HFF) by human NK and LAK cells was studied in inhibition assays. A sample of 60%-deacetylated alpha-D mannose penta-acetate was used as inhibitor that was previously shown to specifically inhibit binding and cytolysis of tumour target cells by human NK and LAK cells. We found now that cytolysis of HCMV-infected HFF was inhibited in a dose-dependent manner showing complete inhibition at concentrations above 640 nmoles/ml mannose acetate. This effect on cytolysis was based on inhibition of conjugate formation between virus-infected cells and CD56+NK and LAK cells. In the presence of mannose acetate (640 nmoles/ml) conjugate formation of virus-infected cells was suppressed down to the level of uninfected cells. The latter showed residual conjugate formation on the basis of adhesive interactions with chemospecifity other than for mannose acetate, which were not capable of triggering cytolytic reactions. Coculturing of target cells with LAK cells appeared to induce expression of additional mannose acetate-specific target sites yielding increases of conjugate formation and cytolysis.

Lack of expression of a 31/33kD surface protein on human colon carcinoma cells is a marker for metastasizing potential.

Two highly metastatic human tumor cell lines, SLU-M1 SLU-M2, were established by in vivo selection in Balb/c-nu/nu mice of SLU-1 xenotransplants derived from an adenocarcinoma of the sigmoid colon. Metastatic spread was screened by transplantation of tissues from various organs of s.c.-tumor-bearing nu/nu mice. A monoclonal antibody, mab ME6H2, prepared against a membrane fraction of HT29 cells, also derived from an adenocarcinoma of the colon, showed high 125I-mab ME6H2 binding only to HT29 and SLU-1 cells, whereas hardly any binding was recorded for SLU-M1 and SLU-M2 cells. All cells of the HT29 and SLU-1 populations exhibited a positive immunofluoresence (IF) but only 1-5% of the SLU-M2 and 10-15% of the SLU-M1 subpopulation. A number of other tumor cell lines did not express the ME6H2 target antigen except for line MCF7, derived from an adenocarcinoma of the breast, which showed an IF positive reaction of 100% of the cells but only 25% of mab binding compared to HT29 and SLU-1 cells. The data indicate that expression of the ME6H2 target antigen is adenocarcinoma-specific and lack of expression is a marker for the metastatic potential of these cells. Mab ME6H2 was rapidly internalized upon binding to viable HT29 cells, resulting in an enhancement of cell growth in vitro and tumor growth in vivo. The mab ME6H2-defined target antigen was isolated from cell lysates by antibody affinity chromatography and was identified as a double band in SDS-PAGE with 31kD and 33kD molecular mass usually present in equal amounts.

Identification of a mannose-acetate-specific 87-kDa receptor responsible for human NK and LAK activity.

Target cell recognition and cytotoxicity of human CD56+ NK and LAK cells is readily inhibited by acetylated mannose. Two respective NK cell receptor candidates were isolated from human leukocyte lysates by mannose acetate affinity chromatography. The 87-kDa receptor showed sequence homologies with lactoferrin and the 59-kDa receptor represented a complex of two Ca-binding proteins MRP-8 and MRP-14 reportedly expressed only by cells of myeloid origin. The 87-kDa receptor exhibited heterogeneity in isoelectric focusing and behaved entirely differently from lactoferrin. Preincubation of tumor target cells with the 87-kDa receptor inhibited competitively target cell recognition and cytotoxicity of human CD56+ NK and LAK cells.

Signal requirement for induction of MHC-unrestricted antitumor cytotoxicity of human T cell CD4+/CD8+ subpopulations.

The role of cosignalling in the generation of MHC-unrestricted cytotoxicity of T cells was studied with CD4+ and CD8+ sub-populations highly purified (> 98%) by immunomagnetic cell sorting using OKT4 mab, Dynal anti-CD4 mab, OKT8 mab, Dynal anti-CD8 mab, and OKT3 mab. Cytotoxicity was determined in 4 h cytotoxicity assays against K562 tumor cells known to lack expression of MHC class 1 and class 2 antigens, thus avoiding interference with anti-CD4- or anti-CD8-mediated signalling. Signal transfer was induced via CD4, CD8, CD3, IL-2 receptor and RG receptor specifically interacting with a plant rhamnogalacturonan (RG). In CD8+ cells, the first signal delivered by the sorting mab (immobilized OKT8 or Dynal anti-CD8 or OKT3) only induced low MHC-unrestricted cytotoxicity but committed the cells to develop largely enhanced cytolytic potential upon stimulation with a second (IL-2 or RG) or third (OKT3, IL-2, RG) signal. The highest cytolytic potential was achieved by cumulative signalling via CD8, CD3, IL-2 receptor and RG receptor. The generation of MHC-unrestricted cytotoxicity of CD8+ cells correlated with increased effector cell/target cell conjugate formation. In CD4+ cells, OKT4 as sorting mab induced very low cytolytic potential, and a moderate committment to IL-2 signals but a stronger one to RG signals, yielding further cytotoxicity enhancement. The highest cytolytic potential was obtained by cumulative signalling via CD4, IL-2 receptor and RG receptor. Dynal anti-CD4 mab was inefficient and OKT3, as sorting mab of CD4+ cells from CD8-depleted PNAC, appeared to block subsequent OKT4-induced generation of MHC-unrestricted cytotoxicity by delivering a negative signal. Immobilized OKT3 as second signal present in cultures of OKT4-sorted CD4+ cells was inefficient. Surprisingly, soluble OKT3 together with IL-2 delivered a positive signal in cultures of OKT4-sorted CD4+ cells.

Enhancement of MHC-unrestricted cytotoxic activity of human CD56+ CD3- natural killer (NK) cells and CD3+ T cells by rhamnogalacturonan: target cell specificity and activity against NK-insensitive targets.

Rhamnogalacturonan-mediated enhancement of MHC-unrestricted cytotoxicity was studied with freshly isolated CD56+CD3- natural killer (NK) cells, interleukin-2 (IL-2)-activated CD56+ lymphokine-activated killer (LAK) cells und IL-2/anti-CD3-activated T cells as effector cells using NK-sensitive and NK-insensitive tumor cells as targets. The rhamnogalacturonan fractions IM, IP, and IQ were prepared from commercially available extracts of Viscum album. The dose/response relation of IM, IP, and IQ demonstrated the presence of various concentrations of cytotoxicity-enhancing compounds in all three fractions that were identified as rhamnogalacturonans by degradation studies with poly-alpha-D-galacturonidase (EC 3.2.1.15) and alpha-1,6-rhamnosidase (EC 3.2.1.40). Specific cytotoxicity of all three effector cell populations as well as the respective rhamnoagalacturonan-mediated cytotoxicity enhancement was readily inhibited in a dose-dependent manner by 60%-deacetylated mannose pentaacetate. Rhamnogalacturonan-mediated enhancement of cytotoxicity of fresh CD56+ NK cells was also observed with four of five NK-insensitive tumor cells as targets, indicating that the effector-cell/tumor-cell bridging activity of rhamnogalacturonans renders NK-insensitive targets susceptible to NK-mediated lysis. Moreover, the rhamnogalacturonan-mediated cytotoxicity enhancement became even more prominent when lymphokine-activated CD56+ LAK and CD3+ T cells were assayed with the NK-insensitive tumor cell targets.

Activation of human monocyte/macrophage cytotoxicity by IL-2/IFN gamma is linked to increased expression of an antitumor receptor with specificity for acetylated mannose.

Spontaneous cytotoxicity of human monocytes (purity: 92-95%) against K562 tumor cells was only observed in 31% healthy donors but, in the presence of rhamnogalacturonan (500 ng/ml), enhanced cytotoxicity was recorded for 79% (n = 14) of the donors. Monocytes activated by culturing with interleukin-2 and/or IFN gamma showed increased antitumor cytotoxicity against K562 tumor cells in 86% (n = 21) of the donors exhibiting additional increases in specific cytotoxicity when the cytotoxicity assays were carried out in the presence of rhamnogalacturonan. Increases of monocyte cytotoxicity achieved by activation with cytokines coincided with increased formation of monocyte/tumor cell conjugates. Similarly, increased monocyte cytotoxicity mediated by rhamnogalacturonan also correlated with increased monocyte/tumor cell conjugate formation most likely due to effector cell/target cell bridging as was originally described for rhamnogalacturonan interacting with CD56+ natural killer or lymphokine-activated killer cells and tumor cells. The chemospecificity of the monocyte-based receptors responsible for cytotoxicity and for monocyte/tumor cell conjugate formation, as well as for their rhamnogalacturonan-mediated enhancements, appears to be identical since all these effects could be inhibited in a dose-dependent manner by partially deacetylated (60%) mannose pentaacetate.

Induction of NK-like activity in T cells by IL-2/anti-CD3 is linked to expression of a new antitumour receptor with specificity for acetylated mannose.

The generation of MHC-unrestricted cytotoxicity of highly enriched human CD3+ T cells (95-99%) by treatment with IL-2 and/or anti-CD3 antibodies was studied. T cells obtained by positive immunomagnetic sorting (anti-CD3) developed comparable specific cytotoxicities against K562 and Daudi cells when cultured with IL-2 and anti-CD3 for 96 h (80% of donors; n = 25). This increase of MHC-unrestricted cytotoxicity correlated fairly well with an increased formation of T cell/tumour cell conjugates. Moreover, simultaneous expression of a rhamnogalacturonan-binding receptor on activated T cells could be demonstrated. Rhamnogalacturonan was reported to enhance cytotoxicity of CD56+ NK cells by effector cell/target cell bridging. Untreated CD3+ cells hardly reacted with rhamnogalacturonan and IL-2-activated T cells showed only a moderate enhancement of cytotoxicity in the presence of rhamnogalacturonan. However, when CD3+ T cells had interacted with anti-CD3 antibodies during cell-sorting or during subsequent culturing with IL-2, enhancement in cytotoxicity and increased formation of lytic effector cell/tumour cell conjugates in the presence of rhamnogalacturonan could be readily demonstrated, indicating a bridging effect analogous to CD56+ NK cells. The conjugate formation of activated T cells with tumour cells as well as the additional rhamnogalacturonan-mediated bridging must be based on the expression of receptors with acetyl mannose specificity, since enhancements of MHC-unrestricted T cell cytotoxicity and conjugate formation were inhibited in a dose-dependent manner when acetylated mannose was present in the assays.

Anti-epidermal growth factor receptor monoclonal antibodies affecting signal transduction.

Monoclonal antibodies prepared against tyrosine phosphorylated epidermal growth factor receptor (EGFR) were tested for their effects on transmembrane signal transduction in A431 tumor cells. Monoclonal antibodies (mab) defined by SDS-sensitive epitopes, i.e., epitopes with conformational specificity, were most effective. Mab 5-125 reacting with a site of the extracellular EGFR domain blocked EGF-binding and cell proliferation in vitro, as well as tumor growth in vivo. However, this mab appeared not to be internalized upon binding to EGFR and did not trigger EGFR autophosphorylation. In contrast, mab 5-D43, also defined by an SDS-sensitive epitope and reacting with an extracellular EGFR site, did not block EGF binding but was readily internalized after binding to EGFR of untreated A431 cells. This mab induced EGFR tyrosine phosphorylation in cell lysates and tyrosine-specific autophosphorylation of insolubilized EGFR immune complexes. Cell growth in vitro was greatly stimulated in the presence of mab 5-D43. Since interaction of mab 5-D43 with EGFR induced most EGF-specific functions, although it did not bind to the EGF-specific site of EGFR, we have to assume that binding of mab 5-D43 to EGFR induced a conformational shift that activated the cytoplasmic EGFR kinase site. On the other hand, activation and/or accessibility of the EGFR kinase site could be blocked by mab 1-594, which is defined by an SDS-insensitive protein epitope of the cytoplasmic EGFR domain. Blocking of the EGFR kinase site by mab 1-594 also abolished EGF-induced tyrosine phosphorylation of endogenous cellular substrates with molecular masses of 145, 97, 85, 37, and 32 kDa, as well as of exogenous substrates such as GAT copolymer.

Mechanism of stimulation of human natural killer cytotoxicity by arabinogalactan from Larix occidentalis.

Cultures of human peripheral blood mononuclear cells (PBMC) as well as cultures of preseparated peripheral non-adherent cells (PNAC) and monocytes showed enhancement of natural killer (NK) cytotoxicity against K562 tumor cells when pretreated with arabinogalactan from Larix occidentalis for 48-72 h. Lack of enhanced responses of PBMC (37% of donors) did not necessarily mean that PNAC and monocyte cultures were also non-responsive to arabinogalactan treatment. Moreover, PBMC, PNAC and monocytes of individual donors could exhibit various responses to arabinogalactan when cultures derived from bleedings after intervals of several months were assayed. Arabinogalactan-mediated enhancement of NK cytotoxicity was not initiated directly but was found to be governed by the cytokine network. Generally, arabinogalactan pretreatment induced an increased release of interferon gamma (IFN gamma), tumor necrosis factor alpha, interleukin-1 beta (IL-1 beta) and IL-6 but only IFN gamma was involved in enhancement of NK cytotoxicity since cytotoxicity enhancement of PBMC and PNAC but not that of monocytes could be blocked when anti-IFN gamma antibodies were present during pretreatment. The presence of anti-IL-2 antibodies completely blocked NK cytotoxicity enhancement of PBMC and only moderately that of PNAC and monocytes. This blocking effect was also observed when no detectable increase of IL-2 release could be recorded. The receptor specificity of arabinogalactan is not well characterized. Initial information obtained from comparative studies indicated that arabinogalactan presumably interacts with a receptor that showed specificity for a NK-cytotoxicity-enhancing oligo-saccharide from Viscum album extracts since the action of both components was not synergistic but rather competitive.

The ontogeny of bovine thymostimulin production in fetal and postnatal age.

The development of thymostimulin production in the bovine fetal thymus was determined, starting at month 2 of gestation until birth. Production of fetal thymostimulin, identified electrophoretically as a peptide with 4-5000 Da, started at month 4 of gestation and achieved its maximum expression three months after birth, followed by a rapid decrease until month 18. Thymus of fetuses from the early gestational phase (2-3 months) yielded no electrophoretically detectable thymostimulin band. Biological activity of the fractions, determined by increased E-rosetting, fairly correspond to the content of the 4-5000 Da peptide moiety.

The CySF-L2 factor from dialysable human leucocyte extract activates natural killer cytotoxicity by induction of interferon gamma.

The mechanism of natural killer (NK) cytotoxicity activation of human peripheral blood mononuclear cells (PBMC) by CySF-L2 was elucidated. CySF-L2 is a cytotoxicity-stimulating factor isolated from dialysable human leucocyte extract, which activates NK cytotoxicity against NK-sensitive and insensitive tumour cells (K562; Daudi; Raji; MOLT4) when preincubated with effector cells for 72 h. CySF-L2-mediated activation was synergistic to interleukin-2(IL-2)-mediated activation of NK cytotoxicity. Induction of interferon gamma (IFN gamma) release was the crucial step during CySF-L2-mediated NK cytotoxicity activation since enhancement of NK activity was completely blocked when anti-IFN gamma antibodies were present during treatment of PBMC. Anti-IFN alpha, anti-TNF alpha (tumour necrosis factor alpha) anti-IL-1 and anti-IL-2 antibodies showed no blocking effect. Analysis of the supernatant culture medium after 72 h incubation of PBMC and their highly purified subpopulations demonstrated that CySF-L2 induced release of IFN gamma from CD3+T cells and CD56+CD3- NK cells and of TNF alpha and prostaglandin E2 from monocytes. CySF-L2 was also capable of activating NK cytotoxicity of highly purified (98%) CD56+CD3- NK cells as well as of monocytes (94% pure). Cell cooperation studies connected with analysis of cytokine release and enhancement of NK cytotoxicity indicated that CySF-L2 might play an essential role in the up and down regulation of NK cytotoxicity by the cytokine network.

Dialysable factors from human leukocyte extracts activating human NK cytotoxicity against tumor cells: identification of factor-producing leukocyte subpopulations.

Dialysable human leukocyte extracts (10 kDa molecular weight cutoff) contain at least three cytotoxicity-stimulating factors CySF-L1, CySF-L2 and CySF-L3, activating natural killer (NK) cytotoxicity of human peripheral blood mononuclear cell (PBMC) against tumor cells when PBMC were pre-incubated with the factors for 72h prior to cytotoxicity assays. The factors could be separated by ion exchange chromatography on Dowex 50WX2. The leukocyte subpopulations producing these factors could be identified: CySF-L1 was detected in granulocyte extracts and T lymphocyte extracts, CySF-L2 in granulocyte extracts and monocyte extracts, CySF-L3 in T lymphocyte extracts and in B lymphocyte/NK cell extracts. All three factors were also present with low concentrations in fresh human plasma. The effector cells activated during pre-incubation of PBMC with the factors were partly identical: CySF-L1 and L2 activated the NK cytotoxicity of monocytes and of NK cells whereas CySF-L3 only stimulated the cytotoxicity of NK cells. All three factors stimulated LAK-like cytotoxicity since the effector cells showed enhanced activity not only against standard NK targets like K562 tumor cells but also against NK-insensitive targets like DAUDI cells and HT29 tumor cells.

Expression of LDL receptor on tumor cells induced by growth factors.

For the determination of LDL receptor expression on living human cells two monoclonal antibodies specific for the extracellular domain of LDL receptor were established using affinity-purified LDL receptor and carrier-conjugated LDL receptor peptide 163-174 as immunizing antigens. The 125I-labeled antibodies were used to quantify increases in LDL receptor expression on human cells grown in the presence of increasing concentrations of various growth factors. Growth factor-mediated increase of LDL receptor expression was entirely different in various cell lines with respect to a distinct growth factor and for different growth factors when tested with one and the same cell line. An increased LDL receptor expression was observed on A431 epidermoid carcinoma cells of the vulva in the presence of epidermal growth factor (EGF) or insulin but not with platelet-derived growth factor (PDGF), on HUV-EC primary endothelial cells in the presence of insulin or PDGF but not with EGF, and on MRC-5 diploid fetal lung cells only in the presence of PDGF. HEP-3B hepatoma cells did not respond to any of the three growth factors essentially maintaining the original level of LDL receptor expression.