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.

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.

Activation of antitumor cytotoxicity of human blood mononuclear cells by a basic factor from dialysable human-leukocyte extract.

Dialysable human leukocyte extract (10 kDa molecular weight cutoff) contained a basic factor stimulating natural killer (NK) cytotoxicity of human peripheral blood mononuclear cells (PBMC) against human K562 tumor cells when PBMC were pre-incubated with the factor for 72h prior to cytotoxicity assays. This cytotoxicity-stimulating factor (CySF-L2) could be enriched by adsorption to ion exchange resin Dowex 50WX2 (H-form) eluting at pH 9.0-9.6 when a pH gradient between pH 3 and pH 10 was used. Further purification was achieved by chromatography on DEAE-Sepharose. The factor has a molecular weight of approximately 1000 Da and is insensitive to protease and exopeptidase treatment but sensitive against treatment with endoglycosidase F. Using immunomagnetic cell sorting, complement-mediated cell depletion and depletion by planning, the cytotoxic effector cells activated during pre-incubation of PBMC with CySF-L2 could be identified as CD16+ CD14+ monocytes/macrophages and as Leu7+ Leu19+ CD16+ CD3- CD8- NK cells.

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.

Mediation of human NK-activity by components in extracts of Viscum album.

Viscum album extracts (Iscador) were investigated for their potency to influence NK cytotoxicity in vitro. In vitro short term cytotoxicity assays (4 h) with human peripheral mononuclear cells (PMNC) and human K 562 tumor cells showed a drastic enhancement of NK cytotoxicity in the presence of V. album extracts. The presence of the V. album components during tumor cell lysis was essential since preincubation of PMNC with V. album extract followed by thorough washing did not lead to enhancement of NK cytotoxicity. One responding effector cell was identified as a member of the large granular lymphocyte (LGL) family carrying both Leu 7 and Leu 11 surface markers. Furthermore, monocytes depleted of LGL, but not differentiated macrophages, showed a weak enhancement of their cytolytic activity in the presence of V. album extract. Fractionation of V. album extracts revealed two active fractions one (C1) with about 3-4000 D and the other (C2) less than 1000 D. Both components enhanced NK cytotoxicity of LGL (Leu 7+, Leu 11+) as well as of monocytes showing enhancing effects also against moderately NK-sensitive tumor cell lines.

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.