IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis.

Natural killer (NK) cells are traditionally considered as innate cells, but recent studies suggest that NK cells can distinguish antigens, and that memory NK cells expand and protect against viral pathogens. Limited information is available about the mechanisms involved in memory-like NK cell expansion, and their role in bacterial infections and vaccine-induced protective immune responses. In the current study, using a mouse model of tuberculosis (TB) infection, we found that interferon-gamma producing CD3-NKp46+CD27+KLRG1+ memory-like NK cells develop during Bacille Calmette-Guérin vaccination, expand, and provide protection against challenge with Mycobacterium tuberculosis (M. tb). Using antibodies, short interfering RNA and gene-deleted mice, we found that expansion of memory-like NK cells depends on interleukin 21 (IL-21). NKp46+CD27+KLRG1+ NK cells expanded in healthy individuals with latent TB infection in an IL-21-dependent manner. Our study provides first evidence that memory-like NK cells survive long term, expansion depends on IL-21, and involved in vaccine-induced protective immunity against a bacterial pathogen.

Interleukin (IL)-21 promotes intestinal IgA response to microbiota.

Commensal microbiota-specific T helper type 17 (Th17) cells are enriched in the intestines, which can convert into T follicular helper (Tfh) in Peyer's patches, and are crucial for production of intestinal immunoglobulin A (IgA) against microbiota; however, the role of Th17 and Tfh cytokines in regulating the mucosal IgA response to enteric microbiota is still not completely known. In this study, we found that intestinal IgA was impaired in mice deficient in interleukin (IL)-17 or IL-21 signaling. IL-21, but not IL-17, is able to augment B-cell differentiation to IgA(+) cells as mediated by transforming growth factor ß1 (TGFß1) and accelerate IgA class switch recombination (CSR). IL-21 and retinoic acid (RA) induce IgA(+) B-cell development and IgA production and drives autocrine TGFß1 production to initiate IgA CSR. Repletion of T-cell-deficient TCRßxδ(-/-) mice with Th17 cells specific for commensal bacterial antigen increased the levels of IgA(+) B cells and IgA production in the intestine, which was blocked by neutralizing IL-21. Thus IL-21 functions to strongly augment IgA production under intestinal environment. Furthermore, IL-21 promotes intestinal B-cell homing through α4ß7 expression, alone or with TGFß and RA. Together, IL-21 from microbiota-specific Th17 and/or Tfh cells contributes to robust intestinal IgA levels by enhancing IgA(+) CSR, IgA production and B-cell trafficking into the intestine.

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on activity of murine thymocytes and peritoneal macrophages.

The purpose of the present study was to investigate properties and mechanism of action of the synthetic adrenocorticotropin (ACTH)-like peptide VKKPGSSVKV, corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. The ACTH-like peptide was shown to act as an immunosuppressive agent in vitro: it inhibits the blast transformation of mouse thymocytes and reduces the spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against Salmonella typhimurium 415 virulent strain bacteria. High affinity receptors for the ACTH-like peptide were found on thymocytes and macrophages and shown to be at the same time the receptors for ACTH. The kinetic characteristics of the ACTH-like peptide and 125I-labeled ACTH (13-24) (ACTH 'address segment') specific binding to the receptors were determined. It was found that the ACTH-like peptide binding to the receptors on target cells is accompanied by an increase in both adenylate cyclase activity and intracellular cAMP content.

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on proliferation of lymphoblastoid cells.

Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10(-11) -10(-7) M inhibits the proliferation of MT-4 cells. Labeled ACTH 'address segment' [(125)I]ACTH (13-24) was used to establish that MT-4 cells express specific receptors for ACTH (K(d) = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [(125)I]ACTH (13-24) for binding to these receptors (K(i1) = 0.38 nM and K(i2) = 0.34 nM).

[Hormone-like activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1]. / Gormonopodobnye svoistva sinteticheskogo dekapeptida, sootvetstvuiushchego adrenokortikotropinopodobnoi posledovatel'nosti immunoglobulina G1 cheloveka.

The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ¿[125I]ACTH-(13-24)¿, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.

Study of immunosuppressive activity of a synthetic decapeptide corresponding to an ACTH-like sequence of human immunoglobulin G1.

The synthetic ACTH-like decapeptide H-Val-Lys-Lys-Pro-Gly- Ser-Ser-Val-Lys-Val-OH, corresponding to amino acid residues 11-20 of the variable part of the human IgG1 heavy chain (referred to as immunocortin) was found to have an immunosuppressive effect on cells in vitro: it inhibits blast transformation of mouse thymocytes and reduces spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against the virulent bacterial strain Salmonella typhimurium 415. Tritium-labeled immunocortin binds with high affinity to ACTH receptors on thymocytes and macrophages (Kd 2. 1 and 2.5 nM, respectively) and activates adenylate cyclase in these cells. Thus, the interaction of immunocortin with the target cell includes the following main steps: binding to the receptor, activation of adenylate cyclase, and elevation of the intracellular content of cAMP.

[L-glutamic acid--a modulator of the physiological status of myeloid series blood cells]. / L-glutaminovaia kislota--moduliator fiziologicheskogo sostoianiia kletok krovi mieloidnogo riada.

The effects of L-glutamic acid on the differentiation of HL-60 and K-562 cell lines and on the expression level of mRNAs encoding IL-1 beta, IL-6, and TNF alpha were studied. It was shown that TNF alpha actively induces the differentiation of these cell lines, whereas L-glutamic acid enhances its effect. Our results indicated that L-glutamic acid modulates the physiological state of the myeloid cell line in blood, in particular, by affecting both the reception and expression of cytokines functionally important for these cells.

[Appearance of glutamate receptors on the surface of HL-60 cells upon differentiation]. / Poiavlenie glutamatnykh retseptorov na poverkhnosti kletok HL-60 pri ikh differentsirovke.

A specific interaction of L-glutamic acid with promyelocytic leukemia HL-60 cells completely differentiated by all-E-retinoic acid (Kd = 0.5 microM) and by plasma membrane fraction from the same cells (Kd = 1 microM) was detected through radioligand analysis and characterized. Quisqualate, a nonlabeled structural analogue of glutamic acid, was found to inhibit competitively the specific binding of L-[3H]glutamic acid to the membranes (Ki = 0.24 microM). The stereospecificity of the binding was demonstrated. These data suggest that specific glutamate receptors appear on the surface of HL-60 cells during their differentiation.

Influence of L-glutamic acid on binding of interleukin-1beta, tumour necrosis factor-alpha and interleukin-6 to HL-60 cells.

To elucidate the mechanism of differentiating activity on L-Glu to HL-60 cells, its influence on binding of human recombinant interleukine-1beta (rHuIL-1beta), tumour necrosis factor-alpha (rHuTNF-alpha) and interleukine-6 (IL-6) by HL-60 cells was studied. It was established, that L-Glu converted the high affinity binding of [125I]rHuIL-1beta (Kd = 0.32 nM) and [125I]rHuIL-6 (Kd = 0.075 nM) to HL-60 cells into low affinity (corresponding Kd values -13.3 and 2.1 nM) at concentration 0.1 microM. The preincubation for an hour of HL-60 cells with 0.1 microM L-Glu was shown to result in an increase of affinity and number of [125I]rHuTNF-alpha binding sites. Thus, L-Glu decreases the sensitivity of the HL-60 cells to IL-1beta and IL-6 and increases TNF-alpha binding at concentration 0.1 microM.

[Effect of L-glutamic acid on the reception of cytokines by HL-60 cells]. / Izuchenie vliianiia L-glutaminovoi kisloty na retseptsiiu tsitokinov kletkami HL-60.

L-Glutamic acid at a concentration of 0.1 microM was found to induce differentiation of the cell line of HL-60 promyelocytic leukemia into granulocytes or neutrophils. The HL-60 cells have no specific glutamate-binding sites, but L-glutamic acid influences the reception of several cytokines by these cells. At a concentration of 0.1 microM, L-glutamic acid completely inhibits the high-affinity binding of 125I-labeled human recombinant interleukin-1 beta (Kd = 0.32 nM) to the HL-60 cells, but does not affect their low-affinity binding (Kd = 13.3 nM) and does not change the total number of the IL-1 beta-binding sites. Preincubation of the HL-60 cells with 0.1 microM of L-glutamic acid increases 2.5 times the number of receptors for 125I-labeled human recombinant tumor necrosis factor beta. These results suggest that L-glutamic acid plays an important role in the differentiation of the blood myeloid cells.

Study of interaction between L-glutamate and human blood lymphocytes.

The presence of specific glutamate receptors on T lymphocytes separated from normal human blood was investigated. Upon study of T lymphocytes interaction with [3H]glutamate the last one was found to bind specifically to these cells, Kd = 2.36 x 10(-7) M. Thirteen unlabelled compounds were tested as potential glutamate competitors. It was shown that only quisqualate (a structural analogue of glutamate) and dipeptides Ala-Glu, Glu-Ala, Glu-Glu competed with [3H]glutamate in binding to a common binding site. The experiments on binding of labelled and unlabelled glutamate bulky conjugates with dextran have demonstrated that both conjugated and free glutamate interacted with the common binding sites on the outer cell membrane. By this means it was shown that T lymphocytes exposed quisqualate-sensitive glutamate receptors on the outer surface of the plasma membrane.

[Interaction of L-glutamic acid with human T-lymphocytes]. / Vzaimodeistvie L-glutaminovoi kisloty s T-limfotsitami cheloveka.

A specific interaction of [3H]Glu with T lymphocytes from the blood of healthy donors (Kd = 0.236 microM) was revealed and described. It was found that unlabeled quisqualate, a structural analogue of L-glutamic acid, and unlabeled dipeptides Ala-Glu, Glu-Ala, and Glu-Glu competitively inhibit the specific binding of [3H]Glu to T lymphocytes (with Ki 0.19, 2.4, 3.4, and 1.2 microM, respectively). Binding experiments with conjugates of labeled and unlabeled glutamic acid with dextran showed that the receptors of [3H]Glu are localized on the outer surface of the plasma membrane of T lymphocytes.

The effect of total saponins from Panax Ginseng C.A. Meyer on the intracellular signalling system in Ehrlich ascites tumor cells.

The mechanisms of action of total saponins from Panax Ginseng C.A. Meyer on the elements of intracellular signalling system in Ehrlich ascites tumor cells were studied. The action of total saponins was compared with the effect of ATP, a classical activator of these cells. Saponins at concentrations of 10(-6)-10(-3)% increased [Ca2+]i, mobilized Ca2+ ions from the endoplasmic reticulum (ER) and activated the influx of Ca2+ to cells. Like ATP, saponins activated the Na+/H+ exchange and Ca(2+)-dependent K+ channels. Of all the parameters, only the activation of Ca2+ influx in cell is directly affected by saponins. The changes in other parameters are connected with nonspecific activation of purinoreceptors. The analysis of the kinetic data suggests that, as distinct from ATP-dependent activation of purinoreceptor, saponins first activate the Ca2+ influx to cells and only then induce the mobilization of Ca2+ from ER.

[Action of triterpene glycosides of the dammarane series and their aglycones on the K+ and H+ ion currents in erythrocytes, induced by the ionophore A23187 and divalent ions]. / Deistvie triterpenovykh glikozidov dammaranovogo riada i ikh glikonov na ionnye potoki K+ i H+ v éritrotsitakh, indutsirovannye ionoforom A23187 i dvukhvalentnymi ionami.

The influence of the dammarane triterpene glycosides Rb1, Rb2, Rg1 and Rf and their aglycones 20(S) protopanaxatriol and 20(S) protopanaxadiol on fluctuation of the K+ and H+ flows in erythrocytes induced by ionophore A23187 and Ca2+ was studied. It was shown that glycoside Rb1 (10 to 50 micrograms/ml) and 20(S) protopanaxatriol (30 micrograms/ml) had a property of inducing fluctuation of the K+ and H+ flows in erythrocytes. In case of Rb1 the system entirely reversed to the initial state whereas in case of the protopanaxatriol use after the fluctuation the system did not reverse to the initial state. Glycosides Rb2 (10 to 30 micrograms/ml), Rg1 (5 to 20 micrograms/ml) and Rf (10 to 100 micrograms/ml) and 20(S) protopanaxadiol (30 micrograms/ml) in the concentrations used did not initiate the fluctuations. The above substances increased the extracellular concentration of K+ and pH without the system reversion to the initial state.

[Mechanism of activation of Ehrlich ascites carcinoma cells using the total fraction of saponins from Korean ginseng]. / Mekhanizm aktivatsii kletok astsitnoi kartsinomy Erlikha obshchei fraktsiei saponinov iz koreiskogo zhen'sheniia.

Molecular mechanisms of action of the total fraction of saponins from Red Korean Ginseng on the elements of the intracellular signalling system of the cells of ascitic Ehrlich carcinoma (AEC) were studied. The action of the total fraction of the saponins on the AEC cells was compared with that of the classic activator of such cells i.e. ATP. It was shown that the action of the total fraction of the saponins was similar to that of ATP. In concentrations of 10(-6) to 10(-3) per cent saponin induced an increase of [Ca2+]j mobilizing Ca2+ from the endoplasmic reticulum (ER) and activating the Ca2+ inlet to the cells. The same as in case with the use of ATP the Ca2+ mobilization from ER was reversible. In comparison to ATP saponin induced higher activation of the Ca2+ inlet to ER and the cells. The same as ATP saponin activated the Na+/H+ exchange and the Ca2+ - dependent K+-channels. Out of all the mention-ed parameters only the activation of the Ca2+ inlet to the cells was probably the direct result of the saponin action. The changes in the other parameters were mediated by nonspecific activation of the purine receptor. The analysis of the kinetic data demonstrated that unlike the ATP-dependent activation of the purine receptor the saponins first of all activated the inlet of Ca2+ to the cells and only after that the mobilization of the latter from ER.