The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L714–L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-d-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1β-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-d-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 μM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.

We have investigated the role of specific components of the thymic stroma during development of CD4-8-T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25+ 44+ precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25+ 44- precursors, develop to the CD4+ 8+ stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25+ 44+ precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to alpha 4 integrin and CD44 interfere with the efficiency with which CD4+ 8+ cells are generated from CD25+ 44+ precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and< or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form.

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We provide evidence that thymocytes receive signals from the thymic microenvironment which regulate the protein kinase C (PKC) signaling pathway. Thus, phorbol 12-myristate 13-acetate (PMA) causes a PKC-dependent down-regulation of CD4 expression and induces apoptosis in isolated thymocytes but has little effect on thymocytes maintained within intact thymic lobes or in reaggregate lobes containing purified thymocytes with either thymic or non-thymic stromal cells. Moreover, compact pellets of thymocytes alone are protected from the effects of PMA. This protection is maintained when the compacted thymocytes are rigorously depleted of MHC class II-expressing cells. We conclude that signals arising from thymocyte-thymocyte contact control the utilization of the PKC cascade. These observations have implications for thymocyte signaling in general as well as for the interpretation of studies carried out on thymocyte suspensions.

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Cells make a malor energetic investment in the synthesis and turriover of inositol 1,3,4,5,6 pentakisphosphate iInsP,l and inositol hexakisphosphate [lnsP,I [refs 1-41 At concentrations of between 1 0 pM and 1 mM, these are the most abundant inositol derivatives in most eukaryotic cells 15 61There is accumulating evidence to suggest that cellular concentrations of InsP, and InsP, may be capable of influencing various cell functions 17-1 11 These include control of neuronal excitability, heart rate and blood pressure, regulation of pituitary function. modulation of the oxygen affinity of haemogiobin in avian erythrocytes, putative roles as antioxidants or antineoplastic agents, and inhibition of enzymes such as an Ins(l.3.4,5IP4 3phosphatase and alkaline phosphatase Interest has recently focussed on possible interactions with arrestin and the clathrin-assembly protein AP-2 However, It remains impossible to interpret these effects in a cellular context, since we do not know whether InsPS and InsP, are located in the appropriate intracellular compartments to exert these effects m situ If either InsP, or InsP, was to have an extracellular role, it should be packaged within vesicles prior to release into the external medium To look for such packaging. we have used several techniques selectively to permeabilize the plasma membrane of the HL60 cell (a human promyelocytic leukaemia cell line that contains a relatively large amount of InsP, and InsP,l without disruption of intracellular organelles within which InsPS and InsP, might be located The degrees to which inositol polyphosphates and intracellular markers were released was then compared InsP, and InsP, were released from cells permeabilized by digitonin, by osmotic lysis or by electroporation in parallel with cytosolic markers ( 6phosphogluconate dehydrogenase and ATPI, and without significant release of the primary granule enzyme ! 3 galactosidase Electroporation was the technique that achieved the most selective permeabilisation of the plasma membrane, and the results obtained with this technique are shown below (Fig 1) During the course of these experiments it was noted that the inclusion of certain ion chelators had modest effects on InsP, and InsP, release from electroporated cells, without effecting the release of inositol or ATP Release I 2 2 x hL.LI i s mlns apart 39.2 i 2.1 36.1 * S.S 65.2 * 1.5 56.8* 1.2 -56.7 i 2.8 73.9 i 9.4 70.6 i 0.6 56.8 f 0.8 66.8 i 0.7 -44.1 i 3.7 SY.3 i 2.3 -57.0 i 1.5 -of InsP, and InsP, was increased significantly by the addition of BAPTA, EGTA or EDTA, but not by the FeiAl-selective agent desferroxamine (Fig 2) 100 90 80 7 0 U 60 m o m m 50 L a4 40 30 20 10 0 Fig. 1 The release of inositol polyphosphates, inositol and cellular markers from HL60 cells permeabilized by high voltage electroporation. /I , ; I 7 InsP,+, i i n o s i t o l I , ; I 7 InsP,+, i i n o s i t o l I I ~ ~---_----0 125 250 375 500 625 750 875 1000 V o l t a g e d i s c h a r g e d ( V / c m ) Data shown are p e r c e n t a~e r of the f o~a l released when 5...