The new ketolide HMR3647 accumulates in the azurophil granules of human polymorphonuclear cells.

HMR3647 is a semisynthetic representative of a new group of drugs, the ketolides, derived from erythromycin A. Since macrolides have been shown to accumulate in human polymorphonuclear cells (PMNs), we have investigated the ability of the molecule HMR3647 to enter human PMNs as well as other cell types, such as peripheral blood mononuclear cells and cell lines of hematopoietic and nonhematopoietic origin. In these experiments, HMR3647 was compared to erythromycin A, azithromycin, clarithromycin, and roxithromycin. Our results show that HMR3647 is specifically trapped in PMNs, where it is concentrated up to 300 times. In addition, it is poorly released by these cells, 80% of the compound remaining cell associated after 2 h in fresh medium. By contrast, it is poorly internalized and quickly released by the other cell types studied. This differs from the results obtained with the macrolide molecules, which behaved similarly in the different cells studied. In addition, subcellular fractionation of PMNs allowed us to identify the intracellular compartment where HMR3647 was trapped. In PMNs, more than 75% of the molecule was recovered in the azurophil granule fraction. Similarly, in NB4 cells differentiated into PMN-like cells, almost 60% of the molecules accumulated in the azurophil granule fraction. In addition, when HMR3647 was added to disrupted PMNs, 63% accumulated in the azurophil granules. Therefore, this study shows that the ketolide HMR3647 specifically accumulates in PMN azurophil granules, thus favoring its delivery to bacteria phagocytosed in these cells.

Evidence for CPP32 activation in the absence of apoptosis during T lymphocyte stimulation.

Cysteine proteases of the interleukin-1beta-converting enzyme family have been implicated in the effector process of apoptosis in several systems. Among these, CPP32 has been shown to be processed to active enzyme at the onset of apoptosis. Here, we show that CPP32 precursor is cleaved into its active form during phytohaemaglutinin A activation of T lymphocytes. Maximal processing is observed between day 3 and day 4 following addition of mitogen and is a transient process. Precursor cleavage is associated with the appearance of a CPP32-like enzymatic activity in cell lysates. At this time in the culture, almost no apoptotic cell and no dead cell can be detected, and T lymphocytes are actively proliferating. CPP32 processing also occurs when lymphocytes are stimulated through an allogeneic primary mixed lymphocyte reaction. Our results suggest that proteolytic activation of CPP32 could be a physiological step during T lymphocyte activation. In addition, these data indicate that CPP32 activation can occur independently of programmed cell death in T lymphocytes.

Evidence for an interleukin-1beta converting enzyme (ICE)-like activity distinct from ICE and responsible for ICE and CPP32 cleavage during apoptosis.

IL-1beta converting enzyme (ICE) and ICE-related proteases (IRPs) have been suggested to play a central role in apoptosis. We report the use of peptidic ICE inhibitors to reassess the role of this enzyme in the apoptosis induced by Fas or TNFalpha receptor ligation in Jurkat cells, U937 cells or monocytes. Our results show that inhibition of IL-1beta processing can be dissociated from inhibition of apoptosis. Indeed, two out of three com-pounds active on ICE are not inhibitory for apoptosis. This shows that ICE is not required for progression in the apoptotic pathway, but that one or several IRPs are necessary. In addition, Western blot analysis of cell lysates shows that both ICE and CPP32 precursors disappear rapidly after apoptosis induction, while ICH-1L precursor remains intact. Concomitant appearance of cleavage products can be visualized for CPP32, but not for ICE, suggesting that the former is proteolytically activated. In addition, this precursor cleavage can be blocked by an ICE inhibitor active on apoptosis. Altogether, our data support the hypothesis that one or several IRPs are necessary for apoptosis and are responsible for ICE and CPP32 cleavage during this process.

Identification of a cysteine protease closely related to interleukin-1 beta-converting enzyme.

The present study describes the identification and molecular cloning of a new member of the interleukin-1 beta-converting enzyme (ICE) family denoted transcript Y (TY). TY is very closely related to both ICE (51% amino acid identity) and a protein named transcript X (TX) (75% amino acid identity) that we recently identified [Faucheu, C., Diu, A., Chan, A.W.E., Blanchet, A.-M., Miossec, C., Hervé, F.,Collard-Dutilleul, V., Gu, Y., Aldape, R., Lippke, J., Rocher, C., Su, M.S.-S., Livingston, D.J., Hercend, T. & Lalanne, J.-L. (1995) EMBO J. 14, 1914-1922]. The amino acids that are implicated in both the ICE catalytic site and in the PI aspartate-binding pocket are conserved in TY. Within the ICE gene family, TY belongs to a subfamily of proteins closely related to the prototype ICE protein. Using transfection experiments into mammalian cells, we demonstrate that TY has protease activity on its own precursor and that this activity is dependent on the presence of a cysteine residue at position 245. However, despite the close similarity between TY and ICE active sites, TY fails to process the interleukin-1 beta precursor. In addition, as already observed for ICE and TX, TY is able to induce apoptosis when overexpressed in COS cells. TY therefore represents a new member of the growing family of apoptosis-inducing ICE-related cysteine proteases.

Effects of Mycoplasma fermentans on the myelomonocytic lineage. Different molecular entities with cytokine-inducing and cytocidal potential.

Mycoplasma fermentans is a mycoplasma species that has been accused of serving as a cofactor of AIDS development. Here, we show that M. fermentans affects the function of human monocytes and myelomonocytic cell lines on at least two different levels. Heat-inactivated mycoplasma particles induce inflammatory cytokines such as IL-1, IL-6, and TNF in monocytes, as well as in THP-1 cells. Moreover, M. fermentans induces IL-10 (but not IL-12) in freshly isolated human monocytes. The cytokine-inducing effect is mediated by lipid-associated molecules. In addition, we have detected a novel biologic activity that resides in the nonlipid-associated protein fraction of M. fermentans (approximate molecular mass: 15 to 30 kDa) and that has a cytocidal effect on nondifferentiated myelomonocytic cell lines (U937 cells, HL-60 cells), as well as on actinomycin-D-sensitized monocytes. Death is accompanied by oligonucleosomal DNA fragmentation and loss of chromosomal DNA. U937 and HL-60 cells fail to produce cytokines and rather undergo cell death in response to heat-inactivated M. fermentans, provided that they are kept in a relatively undifferentiated stage. Whereas the cytokine-inducing activity is a general feature of many mycoplasma species, it appears that only a restricted panel of mycoplasma species exert a cell death-inducing activity. In addition to M. fermentans strains, Mycoplasma penetrans, another hypothetical cofactor of AIDS, possess a cytocidal activity. This does not apply to other mycoplasma species, including pathogenic ones such as Mycoplasma pneumoniae and Ureaplasma urealyticum. The cell death-inducing effect of M. fermentans is not mediated by cytokines and obeys different principles than TNF-alpha-mediated apoptosis. Thus, in contrast to TNF-alpha-induced death, it is not accompanied by a decrease in the mitochondrial transmembrane potential and is not inhibited by preincubation with the antioxidant drug N-acetylcysteine. In synthesis, it appears that certain AIDS-associated mycoplasma species perturb the function and/or generation of cells from the myelomonocytic lineage via several distinct pathways.

A novel human protease similar to the interleukin-1 beta converting enzyme induces apoptosis in transfected cells.

We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin-1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD-2/ICH-1L and CED-3. A computer homology model of TX was constructed based on the X-ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL-1 beta from pro-IL-1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced-3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.