Augmentation of NKT and NK cell-mediated cytotoxicity by peptidoglycan monomer linked with zinc.

BACKGROUND: Peptidoglycan monomer (PGM), which was originally prepared by biosynthesis from culture fluids of penicillin-treated Brevibacterium divaricatum, is an immunostimulator, the activities of which might be improved by addition of zinc (Zn) to the basic molecule. METHODS: To test the possible cytotoxic effects of this new analogue, we analyzed the ability of PGM-Zn and PGM to change the phenotypic profile of hepatic and splenic mononuclear lymphatic cells and to affect the growth of malignant T-cell line YAC-1 and syngeneic thymocytes. RESULTS: Pretreatment of C57BL/6 mice primarily with PGM-Zn over 6 days (10/mg/kg intraperitoneally) significantly enhanced the proportions of NK1.1high+, CD4-CD8-, CD69+, and CD3intermediate/NK1.1+/IL2R-beta+ (NKT) cells in the liver, and major histocompatibility complex class II+, CD69+, and CD8+ cells in the spleen. Both types of cells were highly cytotoxic against YAC-1 and syngeneic thymocytes, increasing the destruction of YAC-1 by 70% on addition of hepatic cells and by 30% on addition of splenic cells. Destruction of thymocytes increased by 10 and 50%, respectively. CONCLUSION: The results point to PGM-Zn as a potent cytotoxicity-inducing agent, which also generates autoreactive NKT cells.


Introduction
Bacterial cell-wall components, such as lipopolysaccharide derived from Gram-negative bacteria and peptidoglycans (PG) isolated from Gram-positive bacterial cell walls, are well-known immunomodulating agents, which participate in the immune reaction generated on antigen or pathogen stimulation. [1][2][3][4][5][6] Both substances in the presence of two soluble serum proteins (i.e. CD14 and lipopolysaccharide binding protein) activate lymphoid targets through the CD14 receptor, which might be viewed as the patterns recognition receptor for various bacterial cell-wall components. 3,4 CD14 then recruits peptidoglycan monomer (PGM) to members of Toll-like receptor (TLR)2, which was identified as an essential component of the PG receptor signaling complex that controls innate responses. [5][6][7] Stimulation of TLR proteins results in the release mainly of pro-inflammatory mediators (interleukin (IL)-1, IL-6, and tumor necrosis factor-a), which in large quantities might promote infection and induce septic shock. 7 In lower doses, however, bacterial products might have immunostimulating and antimetastatic activities linked with the stimulation of both unspecific and specific immune responses. 8 Adjuvant effects have been tried to be explained by stimulation of co-stimulatory activities in antigenpresenting cells, by mimicking of co-stimulatory signals in T cells, or by enhanced uptake of the antigen by dendritic cells that already express costimulatory molecules. 8 Since there is still a paucity of data about the mechanisms leading to inflammatory and immunomodulatory responses induced by Grampositive bacteria, in the present study we tested the effects of PGM, obtained by biosynthesis from culture fluids of penicillin-treated Brevibacterium divaricatum NRRL-2311, 9 and its new analogue PGM-Zn on the innate immune system.
Basically, PGM represents a water-soluble monomeric structural subunit of the polymeric PG, consisting of b1,4-linked N-acetylmuramic acid-N-acetyl-D-glucosamine disaccharide carrying the pentapeptide. Its immunostimulating and antimetastatic activities have been emphasized in many experimental protocols, 10-12 but the precise mechanism of action of PGM is still unknown. Generally, it is held that it uses similar pathways as other Grampositive bacteria. 5,6 This is supported also by our own data, showing that PGM in vitro has a cytokine-stimulating power on human peripheral blood mononuclear cells, leading to release of IL-1, IL-6, and tumor necrosis factor-a in the presence of human serum or soluble CD14. 13 Previously, however, we also reported that addition of zinc might potentiate the immunostimulatory activity of PGM. [14][15][16][17] The new analogue (PGM-Zn) augmented antibody production and isotype switching in mice sensitized with sheep red blood cells, 14 and corrected some immunosuppressive aspects of aging 15 and cholestatic jaundice. 16 We also observed that prolonged treatment of mice with PGM-Zn may result in activation of peritoneal macrophages, which have greater phagocytic activities, as well as the ability to suppress the Con-A-induced blastic transformation of syngeneic splenocytes. 17 Immunomodulatory effects of PGM and PGM-Zn in these experiments correlated with major histocompatibility complex (MHC) class II expression in the liver, 14 in which we also previously noticed an accumulation of zinc during the normal humoral and cellular immune response. 18 Bearing in mind the crucial role of the hepatic natural killer (NK) cells and NKT cells in the control of immune survielance, 19,20 and evidence about the antitumor effects of PGM alone, [10][11][12] in this study we attempted to characterize the effects of PGM-Zn in comparison with the effect of PGM alone on processes of cell-mediated cytotoxicity. For this purpose, we determined the phenotype and function of hepatic and splenic mononuclear lymphatic cells (MNLC) activated in vivo by PGM-Zn or PGM and analyzed their ability to block the growth of the malignant T-cell line YAC-1 and syngeneic thymocytes. The data showed that pretreatment of C57BL/6 mice, particularly with PGM-Zn, might markedly enhance the cytotoxicity of both types of lymphoid cells against NK-sensitive targets, as well as against syngeneic cells. Simultaneous phenotypic analysis of effector cells also suggested that this cytotoxicity in the liver might be linked with activation of NK1.1 + , CD3 intermediate , IL-2Rb + NKT cells, and in the spleen with activation of cytotoxic CD8 + T cells.

Animals
We used inbred, 2-to 3-month old, male C57BL/6 mice. The animals were housed in standard plastic cages, allowed access to standard mouse food pellets and water ad libitum, and exposed to the natural light/dark cycle.

Treatment with PGM-Zn
Immunomodulating agents PGM (GlcNAc-MurNAc-L-Ala-D-iso-Gln-meso-diamminopimelic acid (w-NH2)-D-Ala-D-Ala) and PGM linked with zinc (PGM-Zn), (Pliva, Zagreb, Croatia) were prepared by biosynthesis from the culture fluids of Brevibacterium divaricatum NRRL-2311, as an apyrogenic, watersoluble substance devoid of any toxic effects. 9 The samples used in this study contained less than 0.015 ng of endotoxin/mg of PGM, according to the limulus amebocyte lysate test (Pyrostat Kit; Millipore, Bedford, Massachusetts). In the morning every second day, the mice were injected with PGM-Zn or PGM dissolved in phosphate-buffered saline (PBS) (10 mg/ kg of body weight, intraperitoneally (i.p.)) for 6 days (total dose, 30 mg/kg). Mice in the control groups were treated with the same volume (0.5 ml) of PBS. Two days after the last injection, the animals were sacrificed to isolate the hepatic and splenic MLNC.

Isolation of intrahepatic lymphocytes, splenocytes and thymocytes
Intrahepatic lymphocytes (IHL) were isolated from intact liver after in situ perfusion with PBS, using a modification of the method of Seglen, as we previously described. 21 Resident liver MNLC were isolated by Ficoll-Hypaque density gradient centrifugation (20 min at 800 ´g). A single suspension of spleen cells was prepared in RPMI 1640 medium (Gipco BRL, Basel, Switzerland), after elimination of erythrocytes by lysing solution. The syngeneic thymocytes were prepared in a similar way.

Flow cytometry cytotoxicity assay
Mediators of Inflammation · Vol 11 · 2002 ratios in a final volume of 200 ml at 37°C in a 5% CO 2 atmosphere. After washing in fluorescence-activated cell sorter (FACS) medium, 200 ml of PI (concentration, 10 mg/ml) were added and the percentage of dead cells was measured by flow cytometry. The destroyed cells were counted by detecting cells with both orange (PKH-26) and red (PI) fluorescence.

Statistical analysis
Data were analyzed using the Sigma Plot Scientific Graphing System, Version 1.02. Statistical significance was calculated by Mann-Whitney U test. The differences were considered significant when p < 0.05.

Cytotoxicity of hepatic and splenic MNLC activated by PGM-Zn and PGM against YAC-1
To test the ability of PGM-Zn and PGM-activated intrahepatic and splenic MNC to act against the NKsensitive malignant T-cell line YAC-1, labeled target cells were exposed to killer cells in different killer-totarget ratios (Fig. 4). The data, analyzed after 2 h, showed that PGM-Zn increased, in a dose-dependent manner, the cytotoxicity of both effectors, increasing the destruction of YAC-1 by 70% after addition of hepatic MLNC cells (from 5 to 76% at an effector:target ratio of 50:1, p < 0.01) and by 30% from baseline after the addition of splenic cells (from 10 to 40%, p < 0.05). Although PGM-activated intrahepatic and splenic cells were more cytotoxic than control MNLC (p < 0.01), this cytotoxicity at all ratios was significantly lower than cytotoxicity of PGM-Zn-activated cells (p < 0.01). A similar but less expressed stimulatory effect was obtained with PGM-Zn-activated peripheral blood mononuclear cells, which in the control group showed very small cytotoxic activity against YAC-1 (data not shown).

Cytotoxicity of hepatic and splenic MNLC activated by PGM-Zn and PGM against syngeneic thymocytes
To test the possibility that during the treatment of mice with PGM-Zn or PGM autoreactive clones of NKT cells were generated, hepatic and splenic MNLC were incubated also with labeled syngeneic thymocytes (Fig. 5). The data showed that PGM-Zn augmented the cytotoxicity of hepatic MNLC by 10% (from 10 to 20%, at an effector:target ratio of 50:1). Splenic MNLC, obtained from PGM-Zn-treated mice, however, were more cytotoxic for syngeneic thymocytes than control MNLC, even at a very low effector:target ratio (50% at the ratio 6.25:1, p < 0.01).

Discussion
The data clearly show that PGM and especially PGM-Zn, given in vivo, enhance the antitumor cytotoxicity and autoreactivity of hepatic and splenic MNLC. In the liver, the effect seems to be linked with the increased proportion of activated CD3 int NK1.1 + cells, which after the treatment with PGM-Zn were present in an almost threefold greater proportion than in the control groups of mice (arose from 10.28% to 27.19%). Almost all of them were also IL-2Rb + cells, although PGM-Zn increased also the proportion of the NK1.1 high+ and CD3cell populations (i.e. NK cells). The described phenotype is characteristic for the population of intermediate, NKT cells whose link with the liver is particularly emphasized. 19,20 Supportive elements of their differentiation is IL-7, which is produced by hepatocytes, and IL-12, IL-15 and IL-18, which are produced by Kupffer or endothelial cells. [22][23][24] Although NKT cells phenotypically represent marked heterogeneity, they usually co-express markers that belong to T and NK lineage, and are characterized by a highly restricted TCR repertoire, made of an invariant TCRa chain, Va14-Ja281, asso-ciated with polyclonal Vb8, Vb7, and Vb2 TCR b chains. [22][23][24][25][26][27] It was hypothesized that they represent primordial T cells, which stay at an intermediate phylogenetic position between NK-derived and thymus-derived T cells. 22,23 The current dogma is that this novel lineage of lymphocytes are selected by the nonclassical MHC class I-like molecule, CD1d, which in mice is expressed on dendritic cells, B cells, T cells, macrophages and hepatocytes. 27,28 Unlike the conventional MHC molecules, which bind short peptides in their antigen-binding groove for presentation to either CD4 + or CD8 + T cells bearing ab TCRs, the CD1 molecules are involved in the presentation of lipid and glycolipid antigens. [25][26][27][28][29][30] The hydrophobic part of these antigens most probably binds in the CD1 ligandbinding groove, whereas the polar headgroup of these antigens appears to make direct contact with the TCR and determines specific recognition. 29,30 In such a way, CD1 molecules also display diverse, covalently attached carbohydrates to T cells, that then help B cells to mount an antibody response to polysaccharide antigens. 31 CD1-restricted NKT and T cells therefore play an important role in host defense against microbial infection, but participate also in the recognition of distinct self-antigens, 28 ligands for CD1 molecules might be the glycosylphosphatidylinositol anchors and phosphoinositol mannosides, 33 various metabolites produced by the cells, 34 as well as stress proteins MICA and MICB, 35 supporting the hypothesis that NKT cells represent autoreactive, forbidden, clones of T cells, which after activation might eliminate abnormal self-cells. [22][23][24][25][26] Our data showing increased cytotoxicity of hepatic MNLC after in vivo treatment of mice with PGM-Zn against NK-sensitive and syngeneic targets are, therefore, in agreement with current knowledge of induction and function of hepatic NKT cells in bacterial infection. 22,28,36 We are, however, among the first to show that addition of Zn to PGM might potentiate these activities. Owing to the relevance of zinc for good functioning of the entire immune system, where Zn is acting as a catalyst, structural (zinc fingers) and regulatory ion, 37-41 the observed effects obviously might be obtained by different mechanisms. Enhanced cytotoxicity probably includes the effect of Zn 2+ on the level of 'NK cell immune synapse', where Zn modulates specifically the negative signal transmitted to the NK cells, after binding of killer cell immunoglobulin-like receptors to MHC class I proteins. 42,44 However, owing to the possibility that PGM-Zn acts as superantigen, which markedly enhances the activities of T and B lymphocytes, it could be hypothesized that some changes occurred also at the level of MHC class II molecules, outside the peptidebinding groove, since co-ordination of Zn 2+ is required for high binding of superantigen to this place. 44 Besides numerous other possibilities, [37][38][39][40][41] we would like also to emphasize the crucial role of zinc for the regulation of apoptosis and cell proliferation, 45,46 because this evidence permits one to speculate that PGM-Zn was directly involved in the apoptotic process and CD1 presentation of selfantigens. Namely, during the infection, activated monocytes and other cells undergo spontaneous apoptosis, which involves Fas/Fas ligand interactions. [47][48][49] Moreover, since binding of bacterial toxins and bacterial lipoproteins through TLR2 signals for apoptosis through the myeloid differentiation factor 88 via a pathway involving Fas-associated death domain protein and caspase 8, as well as for cytokine production, 50 there is a possibility that some of the PGM-Zn-induced modifications of cell-mediated cytotoxicity occurred on the level of Toll-like receptor-2, which is a ligand also for peptidoglycans, released from Gram-positive bacteria. 1-6 Furthermore, since it is known that the intracellular content released from dying cells may stimulate the generation of autoreactive NKT, 24,27,32,[33][34][35] as well as adaptive immune responses, 51 it could be speculated that, in such a way, PGM-Zn has augmented also the cytotoxicity of splenic MNLC to NK-sensitive and syngeneic targets (Fig. 5). The recent finding of a high-speed communication network between the NKT cells, generated in vivo after treatment with Alpha-GalCer, and the adaptive immune systems 52 seems to support this conclusion.
Taken together, although the further characteristics of the effects of PGM-Zn on innate immunity remain to be elucidated, our data provide the first evidence that addition of zinc to PGM markedly enhances the generation of hepatic NKT cells and increases the cytotoxic potential of hepatic and splenic MNLC against NK-sensitive and syngeneic targets.