Differential microRNA expression between decidual and peripheral blood natural killer cells in early pregnancy.

STUDY QUESTION: Have decidual natural killer (dNK) cells a different microRNA (miRNA or miR) expression pattern compared to NK cells circulating in the peripheral blood (pb) of healthy pregnant women in the first trimester of gestation? SUMMARY ANSWER: dNK cells have a unique miRNA profile, showing exclusive expression of a set of miRNAs and significant up- or down-regulation of most of the miRNAs shared with pbNK cells. WHAT IS KNOWN ALREADY: dNK cells differ from pbNK cells both phenotypically and functionally, and their origin is still debated. Many studies have indicated that miRNAs regulate several important aspects of NK cell biology, such as development, activation and effector functions. STUDY DESIGN, SIZE, DURATION: Decidua basalis and peripheral blood specimens were collected from women (n = 7) undergoing voluntary termination of gestation in the first trimester of pregnancy. dNK and pbNK cells were then highly purified by cell sorting. PARTICIPANTS/MATERIALS, SETTING, METHODS: miRNAs expression was analysed by quantitative RT-PCR (qRT-PCR)-based arrays using RNA purified from freshly isolated and highly purified pbNK and dNK cells. Results from arrays were validated by qRT-PCR assays. The bioinformatics tool ingenuity pathway analysis (IPA) was applied to determine the cellular network targeted by validated miRNAs and the correlated biological functions. MAIN RESULTS AND THE ROLE OF CHANCE: Herein, we identified the most differentially expressed miRNAs in NK cells isolated from peripheral blood and uterine decidua of pregnant women. We found that 36 miRNAs were expressed only in dNK cells and two miRNAs only in pbNK cells. Moreover, 48 miRNAs were commonly expressed by both NK cell preparations although at different levels: 28 were upregulated in dNK cells, while 15 were downregulated compared to pbNK cells. Validation of a selected set (n = 11) of these miRNAs confirmed the differential expression of nine miRNAs: miR-10b and miR-214 expressed only in dNK cells and miR-200a-3p expressed only in pbNK cells; miR-130b-3p, miR-125a-5p, miR-212-3p and miR-454 were upregulated while miR-210-3p and miR-132 were downregulated in dNK cells compared to pbNK cells. IPA network analysis identified a single network connecting all the miRNAs as well as their significant involvement in several classes of functions: 'Organismal injury, Reproductive system disease, Inflammatory disease' and 'Cellular development'. These miRNAs target molecules such as argonaute 2, tumour protein p53, insulin and other genes that belong to the same network and significantly influence cell differentiation and pregnancy. LIMITATIONS, REASONS FOR CAUTION: In the present study, the cellular network and biological functions modulated by miRNAs differentially expressed in dNK and pbNK cells were identified by IPA considering only molecules and relationships that were with confidence 'experimentally observed' in leucocytes. The decidual and pbNK cells that were analysed here are a heterogeneous population and further study will help to disentangle whether there are differences in miRNA production by the different subsets of NK cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study describing a different miRNA expression profile in dNK cells compared to matched pbNK cells during the first trimester of pregnancy. Our findings improved the body of knowledge on dNK cell biology and strongly suggest further investigation into the roles of miRNAs that are differentially expressed in human dNK compared to pbNK cells. Our results suggest that specific miRNAs can modulate dNK cell origin and functions, highlighting a potential role of this miRNA signature in human development and diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Istituto Pasteur, Fondazione Cenci Bolognetti, the European NoE EMBIC within FP6 (Contract number LSHN-CT-2004-512040), Istituto Italiano di Tecnologia, and Ministero dell'Istruzione, dell'Università e della Ricerca (Ricerche Universitarie), and from Università Politecnica delle Marche. There are no conflicts of interest to declare.

Lavandula angustifolia Mill. Essential Oil Exerts Antibacterial and Anti-Inflammatory Effect in Macrophage Mediated Immune Response to Staphylococcus aureus.

Different studies described the antibacterial properties of Lavandula angustifolia (Mill.) essential oil and its anti-inflammatory effects. Besides, no data exist on its ability to activate human macrophages during the innate response against Staphylococcus aureus. The discovery of promising regulators of macrophage-mediated inflammatory response, without side effects, could be useful for the prevention of, or as therapeutic remedy for, various inflammation-mediated diseases. This study investigated, by transcriptional analysis, how a L. angustifolia essential oil treatment influences the macrophage response to Staphylococcus aureus infection. The results showed that the treatment increases the phagocytic rate and stimulates the containment of intracellular bacterial replication by macrophages. Our data showed that this stimulation is coupled with expression of genes involved in reactive oxygen species production (i.e., CYBB and NCF4). Moreover, the essential oil treatment balanced the inflammatory signaling induced by S. aureus by repressing the principal pro-inflammatory cytokines and their receptors and inducing the heme oxygenase-1 gene transcription. These data showed that the L. angustifolia essential oil can stimulate the human innate macrophage response to a bacterium which is responsible for one of the most important nosocomial infection and might suggest the potential development of this plant extract as an anti-inflammatory and immune regulatory coadjutant drug.

Detection of new genetic profiles and allelic variants in improperly classified grapevine accessions.

Thirty-seven grapevine accessions, collected in Central Italy, were characterized by morphological and genetic analysis, according to guidelines developed by European Union programs of grapevine research and standardization. Traditional denominations of some sampled varieties were revealed to be incorrect; moreover, 10 synonymies and 12 homonymies were recognized. Ampelographic and ampelometric measurements of leaf characters were performed. These data generated a phenotypic similarity matrix and a relative diagram showing morphological differences between specimens. Many samples presented different morphology even in the presence of the same genotype, probably as a result of various environmental pressures. Grapevines were typed by 12 microsatellite loci and then compared with the CRA-VIT genetic resource database. Twenty-five SSR profiles were clearly identified as well-known cultivars, while nine genotypes did not find a direct correspondence: these samples could represent putative new autochthonous Latial Vitis vinifera cultivars or hybrid varieties. The genetic approach also detected three new (169 and 173 in VVMD27 locus; 179 in ISV2 locus) and seven rare allelic variants. Plant sample classification by oral history, morphological observations, and molecular results were compared and discussed. Scions of samples were planted in the Botanic Garden of the University of Rome "Tor Vergata", to preserve grapevine biodiversity and to protect possible new autochthonous varieties.

Tetracycline accumulates in Iberis sempervirens L. through apoplastic transport inducing oxidative stress and growth inhibition.

Environmental antibiotic contamination is due mainly to improper and illegal disposal of these molecules that, yet pharmacologically active, are excreted by humans and animals. These compounds contaminate soil, water and plants. Many studies have reported the bioaccumulation of antibiotics in plants and their negative effects on photosynthesis, cell growth and oxidative balance. Therefore, the principal objective of this paper was the study of antibiotic accumulation sites in plants and its uptake modality. Iberis sempervirens L., grown in soil and in agar in the presence or absence of tetracycline, were used as a model system. Using confocal and transmission electron microscopy, we demonstrated that tetracycline was absorbed and propagated in plants through apoplastic transport and also accumulated in intercellular spaces. Tetracycline was rarely detected inside cells (in cytoplasm and mitochondria where, coherent to its pharmacological activity, it probably affected ribosomes), except in stomata. Moreover, we verified and clarified further the phytotoxic effects of tetracycline on plants. We observed that the antibiotic induced a large reduction in plant growth and development and inhibition of photosynthetic activity. As tetracycline may lead to oxidative stress in plants, plant cells tried to balance this disequilibrium by increasing the amount and activity of some endogenous enzyme antioxidant agents (superoxide dismutase 1 and catalase) and levels of antiradical secondary metabolites.

Characterization of biofilm-forming cyanobacteria for biomass and lipid production.

AIMS: This work reports on one of the first attempts to use biofilm-forming cyanobacteria for biomass and lipid production. METHODS AND RESULTS: Three isolates of filamentous cyanobacteria were obtained from biofilms at different Italian sites and characterized by a polyphasic approach, involving microscopic observations, ecology and genetic diversity (studying the 16S rRNA gene). The isolates were grown in batch systems and in a semi-continuous flow incubator, specifically designed for biofilms development. Culture system affected biomass and lipid production, but did not influence the fatty acid profile. The composition of fatty acids was mainly palmitic acid (>50%) and less amounts of other saturated and monounsaturated fatty acids. Only two isolates contained two polyunsaturated fatty acids. CONCLUSIONS: Data obtained from the flow-lane incubator system would support a more economical and sustainable use of the benthic micro-organisms for biomass production. The produced lipids contained fatty acids suitable for a high-quality biodiesel production, showing high proportions of saturated and monounsaturated fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: Data seem promising when taking into account the savings in cost and time derived from easy procedures for biomass harvesting, especially when being able to obtain the co-production of other valuable by-products.

PIM1 kinase is destabilized by ribosomal stress causing inhibition of cell cycle progression.

PIM1 is a constitutively active serine/threonine kinase regulated by cytokines, growth factors and hormones. It has been implicated in the control of cell cycle progression and apoptosis and its overexpression has been associated with various kinds of lymphoid and hematopoietic malignancies. The activity of PIM1 is dependent on the phosphorylation of several targets involved in transcription, cell cycle and apoptosis. We have recently observed that PIM1 interacts with ribosomal protein (RP)S19 and cosediments with ribosomes. Defects in ribosome synthesis (ribosomal stress) have been shown to activate a p53-dependent growth arrest response. To investigate if PIM1 could have a role in the response to ribosomal stress, we induced ribosome synthesis alterations in TF-1 and K562 erythroid cell lines. We found that RP deficiency, induced by RNA interference or treatment with inhibitor of nucleolar functions, causes a drastic destabilization of PIM1. The lower level of PIM1 induces an increase in the cell cycle inhibitor p27(Kip1) and blocks cell proliferation even in the absence of p53. Notably, restoring PIM1 level by transfection causes a recovery of cell growth. Our data indicate that PIM1 may act as a sensor for ribosomal stress independently of or in concert with the known p53-dependent mechanisms.

Integrin pattern and effect on contraction in cultured testicular peritubular myoid cells.

PROBLEM: Neither the integrin pattern nor the biological functions of integrins have been extensively documented in human cultured testicular peritubular myoid cells (TPMC). The integrin pattern and the presence of some proteins of the immunoglobulin superfamily on human TPMC as well as the role of integrins in TPMC contraction were examined. METHOD OF STUDY: Integrin expression was evaluated by immunofluorescence and FACS analysis. To assess the role of integrin in TPMC contraction, human and rat cells were added to a collagen gel system and exposed to contractile stimuli. RESULTS: The immunofluorescence and cytofluorimetric analysis showed that human cultured TPMC express alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, and beta4 integrin subunits, and significant amounts of intercellular adhesion molecule-1 (ICAM-1), whereas they do not present alpha4, beta2, beta7 subunits, nor intercellular adhesion molecule-2 (ICAM-2) and neural cell adhesion molecule (NCAM). The preincubation of human cells with an anti-beta1 mAb and of rat cells with a polyclonal anti-beta1 antibody inhibited TPMC contraction induced by different contractile stimuli. CONCLUSION: Our investigation documented a broad integrin pattern on human cultured TPMC as well as a role for integrins in human and rat TPMC contraction.

RAC1/P38 MAPK signaling pathway controls beta1 integrin-induced interleukin-8 production in human natural killer cells.

The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that beta1 integrin ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the beta1 integrin-mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by beta1 integrins.

Cutting edge: functional role for proline-rich tyrosine kinase 2 in NK cell-mediated natural cytotoxicity.

Protein tyrosine kinase activation is one of the first biochemical events in the signaling pathway leading to activation of NK cell cytolytic machinery. Here we investigated whether proline-rich tyrosine kinase 2 (Pyk2), the nonreceptor protein tyrosine kinase belonging to the focal adhesion kinase family, could play a role in NK cell-mediated cytotoxicity. Our results demonstrate that binding of NK cells to sensitive target cells or ligation of beta2 integrins results in a rapid induction of Pyk2 phosphorylation and activation. By contrast, no detectable Pyk2 tyrosine phosphorylation is found upon CD16 stimulation mediated by either mAb or interaction with Ab-coated P815 cells. A functional role for Pyk2 in natural but not Ab-mediated cytotoxicity was demonstrated by the use of recombinant vaccinia viruses encoding the kinase dead mutant of Pyk2. Finally, we provide evidence that Pyk2 is involved in the beta2 integrin-triggered extracellular signal-regulated kinase activation, supporting the hypothesis that Pyk2 plays a role in the natural cytotoxicity by controlling extracellular signal-regulated kinase activation.

Evidence for alphavbeta3 and alphavbeta5 integrin-like vitronectin (VN) receptors in Candida albicans and their involvement in yeast cell adhesion to VN.

The expression of integrin vitronectin (VN) receptors on Candida albicans yeasts and their involvement in the adhesion to VN were investigated. By immunofluorescence and cytofluorimetric analysis, several antibodies directed against human alphav, beta3, beta5, alphavbeta3, or alphavbeta5 integrin positively stained C. albicans yeasts. Biochemical analysis on yeast lysates with anti-human alphav, beta3, or beta5 antibody revealed molecular species of 130, 110, 100, and 84 kDa. The 130-kDa band was identified as alphav, whereas the doublet of 110/100 kDa and the 84-kDa band likely correspond to the beta3 and beta5 subunits, respectively. Some 48%-54% of Candida yeasts specifically adhered to VN, and this binding was strongly inhibited by anti-human alphav, beta3, alphavbeta3, and alphavbeta5 antibodies and by RGD- but not RGE-containing peptides. In addition, VN inhibited C. albicans adherence to a human endothelial cell line. Thus, C. albicans in the yeast phase expresses VN receptors antigenically related to the vertebrate alphavbeta3 and alphavbeta5 integrins, which mediate its adhesion to VN.

Beta 1 integrin cross-linking inhibits CD16-induced phospholipase D and secretory phospholipase A2 activity and granule exocytosis in human NK cells: role of phospholipase D in CD16-triggered degranulation.

Recent data indicate that integrin-generated signals can modulate different receptor-stimulated cell functions in both a positive (costimulation) and a negative (inhibition) fashion. Here we investigated the ability of beta 1 integrins, namely alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors, to modulate CD16-triggered phospholipase activation in human NK cells. beta 1 integrin simultaneous cross-linking selectively inhibited CD16-induced phospholipase D (PLD) activation, without affecting either phosphatidylinositol-phospholipase C or cytosolic phospholipase A2 (PLA2) enzymatic activity. CD16-induced secretory PLA2 (sPLA2) protein release as well as its enzymatic activity in both cell-associated and soluble forms were also found to be inhibited upon beta 1 integrin coengagement. The similar effects exerted by specific PLD pharmacological inhibitors (2,3-diphosphoglycerate, ethanol) suggest that in our experimental system, sPLA2 secretion and activation are under the control of a PLD-dependent pathway. By using pharmacological inhibitors (2,3-diphosphoglycerate, wortmannin, ethanol) we also demonstrated that PLD activation is an important step in the CD16-triggered signaling cascade that leads to NK cytotoxic granule exocytosis. Consistent with these findings, fibronectin receptor engagement, by either mAbs or natural ligands, resulted in a selective inhibition of CD16-triggered, but not of PMA/ionomycin-induced, degranulation that was reversed by the exogenous addition of purified PLD from Streptomyces chromofuscus.

Integrin-mediated ras-extracellular regulated kinase (ERK) signaling regulates interferon gamma production in human natural killer cells.

Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that beta1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc-growth factor receptor-bound protein 2 (Grb2) and Shc-proline-rich tyrosine kinase 2-Grb2 complexes are the receptor-proximal events accompanying the beta1 integrin-mediated Ras activation. In addition, we demonstrate that ligation of beta1 integrins results in the stimulation of interferon gamma (IFN-gamma) production, which is under the control of extracellular signal-regulated kinase 2 activation. Overall, our data indicate that beta1 integrins, by delivering signals capable of triggering IFN-gamma production, may function as NK-activating receptors.

CD16-mediated activation of phosphatidylinositol-3 kinase (PI-3K) in human NK cells involves tyrosine phosphorylation of Cbl and its association with Grb2, Shc, pp36 and p85 PI-3K subunit.

Phosphatidylinositol 3-kinase (Pl-3K) plays a key role in several cellular processes, including mitogenesis, apoptosis, actin reorganization and vesicular trafficking. The molecular events involved in its activation have not been fully elucidated and several reports indicate that a key event for enzyme activation is the interaction of the SH2 domains of the p85 regulatory subunit of Pl-3K with tyrosine-phosphorylated proteins. In this study, we investigated the involvement of the product of the proto-oncogene c-Cbl in the activation of Pl-3K triggered by CD16 in human NK cells and the possible mechanisms leading to Pl-3K recruitment to the plasma membrane. Our results indicate that stimulation of NK cells through CD16 results in a rapid tyrosine phosphorylation of Cbl, which is constitutively associated with Grb2 and forms an activation-dependent complex with the p85 subunit of Pl-3K. In addition, we detected the presence of the Grb2-associated tyrosine-phosphorylated p36 and Shc proteins in anti-Cbl and anti-p85 immunoprecipitates from CD16-stimulated NK cell lysates. Upon CD16 stimulation, Pl-3K activity was found associated with Cbl and to a lesser extent with Grb2 and Shc as well as with the zeta chain of the CD16 receptor complex. Overall these results suggest that the formation of a complex containing either Shc or pp36 associated with Grb2, Cbl and the p85 subunit of Pl-3K is one of the major mechanisms which might couple CD16 to the Pl-3K pathway in NK cells.

Proline-rich tyrosine kinase-2 activation by beta 1 integrin fibronectin receptor cross-linking and association with paxillin in human natural killer cells.

Recent evidence indicates that integrin ligation results in activation of focal adhesion kinase (pp125FAK), the prototype of a new subfamily of nonreceptor protein tyrosine kinase (PTK), including FAKB and the proline-rich tyrosine kinase 2 (PYK-2), also termed cell adhesion kinase-beta or related adhesion focal tyrosine kinase. We have previously shown that cross-linking of alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors on human NK cells stimulates tyrosine phosphorylation of two proteins migrating at 105 and 115 kDa. Here we report that cross-linking of beta 1 integrins on human NK cells stimulates tyrosine phosphorylation and PTK activity of PYK-2. PYK-2 tyrosine phosphorylation was maximal at 1 min and started to decline 20 min after stimulation. Engagement of alpha 4 beta 1 and alpha 5 beta 1 either with specific mAbs or after cell adhesion to fibronectin or its 120- and 40-kDa fragments also triggered PYK-2 tyrosine phosphorylation. Stimulation of PYK-2 tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by EGTA, indicating that PYK-2 tyrosine phosphorylation is PTK, but not calcium, dependent. We also demonstrate that PYK-2 is constitutively associated with paxillin, which undergoes tyrosine phosphorylation with the same kinetics of PYK-2 upon beta 1 integrin ligation.

CD16 cross-linking induces both secretory and extracellular signal-regulated kinase (ERK)-dependent cytosolic phospholipase A2 (PLA2) activity in human natural killer cells: involvement of ERK, but not PLA2, in CD16-triggered granule exocytosis.

The phospholipase A2 (PLA2) enzymes play a central role in diverse cellular processes including phospholipid digestion and metabolism, host defense, and cell signaling. We investigated the ability of CD16 clustering to trigger PLA2 and extracellular signal-regulated kinase (ERK) activation in human NK cells, as well as their possible involvement in CD16-stimulated degranulation. Both secretory (sPLA2) and cytosolic (cPLA2) PLA2 were rapidly activated upon CD16 cross-linking; sPLA2 was found in the supernatant and also in a cell-associated form. cPLA2 activation was controlled by the ERK pathway as indicated by the close correlation between their kinetics of activation and by the ability of the specific MEK inhibitor, PD 098059, to abolish cPLA2 activation. CD16 stimulation also resulted in the generation of platelet-activating factor (PAF) and leukotrienes; both phospholipases contributed to their biosynthesis. Using the pharmacologic inhibitors AACOCF3, p-bromophenacyl bromide (pBPB), and PD 098059, which specifically inhibit cPLA2, sPLA2, and MEK, respectively, we demonstrated that the ERK signaling pathway, but not cytosolic or secretory PLA2, is required for CD16-triggered granule release.

Functional analysis of alpha 1 beta 1 integrin in human natural killer cells.

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

beta 1-Integrin expression and function in human bladder cancer cells: modulation by TNF alpha.

Cytofluorimetric and biochemical analysis in two different grade human bladder cancer cell lines showed that G3 EJ cells exhibited higher levels of alpha5beta1 and alpha6beta1 heterodimers, and the G2 RT112 cell line higher levels of alpha2beta1. Alpha6/beta4 receptor was detected only in RT112 cells. Adhesion assays with extracellular matrix proteins indicated that both cells bound to fibronectin, laminin and collagen 1, the adhesive properties being related to the integrin profile. Inhibition tests revealed that alpha5beta1 mediated adhesion to fibronectin, alpha3beta1 and alpha6beta1 to laminin, and that alpha2beta1 was the main mediator of adhesion to collagen I in both cell lines. In EJ but not in RT112 cells, tumor necrosis factor-alpha induced the upregulation of alpha2, which mediated increased adhesion to collagen I. The different effects of TNFalpha on the two cell lines were not attributable to differences in tumor necrosis factor responsiveness, as both cells expressed comparable levels of tumor necrosis factor receptor-1 and the tumor necrosis factor-inducible intercellular adhesion molecule-1.

Interaction of natural killer cells with extracellular matrix induces early intracellular signalling events and enhances cytotoxic functions.

NK cells are endowed with a wide array of adhesion molecules which mediate their interaction with endothelium and extracellular matrix components. We have shown that cross-linking of beta 1 integrins and CD44 on human NK cells induces a signal transduction pathway involving both tyrosine kinase activation and the modulation of intracellular calcium levels. Our studies have also demonstrated the ability of beta 1 integrins and CD44 to upregulate both the spontaneous and the CD16-triggered cytotoxic activity of human NK cells. Although the molecular mechanisms responsible for this costimulatory activity have not been defined yet, our studies indicate that the simultaneous cross-linking of beta 1 integrins and CD44 results in a synergistic effect on CD16-mediated enhancement of intracellular free calcium concentration, and suggest that this may be relevant for the cooperation observed.