Roberto Testi

The CD69 receptor: a multipurpose cell-surface trigger for hematopoietic cells

CD69 was initially described as being restricted to recently activated lymphoid cells, but is now known to be expressed on the surface of all hematopoietically derived leukocytes. Crosslinking of CD69 generates intracellular signals in all cell lineages studied, both mouse and human, and results in a variety of cellular end responses. Since a specific ligand has not yet been identified, a definite functional identity for CD69 is still missing. However, as discussed here by Roberto Testi and colleagues, the broad expression of CD69 and its conserved ability to generate intracellular signals suggests a general role for the CD69 receptor in the biology of hematopoietic cells.

9-O-acetyl GD3 protects tumor cells from apoptosis

The ganglioside GD3 (Neu5Acα8Neu5Acα3Galβ4GlcCer) is an intracellular lipid messenger that induces apoptosis by targeting mitochondria in various cell types. GD3 can also promote apoptosis when externally added to cells. Previous studies showed that the proapoptotic effects of GD3 can be counteracted by 9‐O‐acetylation. To determine whether 9‐O‐acetyl GD3 (acGD3) has a general antiapoptotic potential, the apoptosis‐sensitive Jurkat cell line and an apoptosis‐sensitive variant of the cell line Molt‐4 were preincubated with micromolar concentrations of acGD3 and then treated with inducers of apoptosis. A reduced apoptotic index and an increased cell viability were observed. On the other hand, when the Jurkat cells were treated with GD3 for extended periods of time, a population was selected that was resistant to apoptosis induction by N‐acetyl sphingosine as well as by the anti‐leukemic drug daunorubicin. Comparative analysis of gangliosides revealed the formation of acGD3 in the resistant Jurkat cells that was not found in the apoptosis‐sensitive cells. Conversely, exposing the acGD3 positive and apoptosis‐resistant cell line Molt‐4 to the O‐deacetylating activity of salicylate resulted in a complete disappearance of acGD3 and an enhanced sensitivity to N‐acetyl sphingosine‐mediated apoptosis. Formation of acGD3 might thus represent a new mechanism how tumor cells can escape apoptosis.

Proliferative effects of 12-O-Tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL)☆

The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c(PKC), and to the Ca2+ ionophores A23817 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A23817. TPA alone (1-20 ng/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 ng/ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fc gamma receptors (CD16) on LGL and the induction of the expression of IL-2 (CD25) and transferrin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca2+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tac antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum.

Sequential metabolic events and morphological changes during in vivo large granular lymphocyte activation and proliferation

Interferon (IFN) and IFN inducers are known to boost natural killer (NK) activity in vivo and in vitro. In vivo enhancement of NK activity results from activation of preexisting NK cells as well as from an increased number of large granular lymphocytes (LGL), with a portion of them undergoing cell division. Our study was addressed to analyze the sequence of metabolic events occurring within the LGL population of Fischer rats treated with poly(I:C), as an IFN inducer. The increase in cytotoxic activity and LGL number in the peripheral blood already reached maximal levels by 12 hr after poly(I:C) injection, remained on a plateau 24 to 48 hr later, then slightly decreased on Day 4, and returned to control levels by Day 6. A similar kinetics was observed for RNA synthesis. In contrast DNA synthesis first increased at 24 hr, peaked at 48 hr, then decreased on Day 4, and was not detectable on Day 6. Percoll fractionation resulted in 92-97% of LGL in fraction 1, and cells in this fraction accounted for the increase of cytotoxicity as well as for newly synthesized RNA and DNA. However, LGL recovered on Day 1 or 2 after poly(I:C) stimulation displayed quite heterogeneous morphology, and a number of mitotic configurations were seen on Day 2 within the LGL population. Our results indicate that the boosting of NK activity by poly(I:C) is always associated with an increase in LGL numbers, the enhanced lytic capacity is associated in vivo with new RNA synthesis by the NK cells, and only in a later phase NK cell proliferation may account for the increase in LGL numbers.

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.

Persistent In Vivo Activation and Transient Anergy to TCR/CD3 Stimulation of Normal Human Intestinal Lymphocytes

Intestinal lymphocytes represent a unique lymphoid compartment heavily and constantly exposed to antigens. Continuous antigen challenge however, does not normally result in an inflamatory tissue damage, suggesting that cell activation mechanisms are tightly controlled in gut mucosal lymphocytes. Normal intestinal lymphocytes have been found in fact to express in vivo some degree of phenotypic activation, yet little if any evidence of in vivo proliferation is detectable. Accordingly, TCR/CD3 mediated in vitro proliferative signals are consistently inefficient. This peculiar behavior renders the intestinal immune system an ideal model to unravel in vivo operating mechanisms of suppression and tolerance induction.1

LAMININ PRODUCTION BY NATURAL KILLER CELLS

ABSTRACT Here we report using different approaches that NK cells synthesize and secrete laminin, a glycoprotein of extracellular matrix.