hormonally regulated. That a proper balance between cell death and survival-promoting proteins is critical to achieve Transgenic mice expressing high levels of the BclxL or this physiological apoptotic wave at an early stage of Bcl2 proteins in the male germinal cells show a highly testicular germinal cell differentiation is substantiated by abnormal adult spermatogenesis accompanied by the finding of a comparable syndrome in mice defective sterility. This appears to result from the prevention of in the bax gene (Knudson et al., 1995). This early an early and massive wave of apoptosis in the testis, apoptotic wave of germ cells during establishment of which occurs among germinal cells during the first spermatogenesis may be required to maintain a proper round of spermatogenesis. In contrast, sporadic cell number ratio between maturing germ cells and Sertoli apoptosis among spermatogonia, which occurs in nor-cells. An intricate relationship involving a constant modumal adult testis, is not prevented in adult transgenic lation of activity and exchange of differentiation signals mice. The physiological early apoptotic wave in the indeed exists between these two cell types (reviewed in testis is coincident, in timing and localization, with a Jegou, 1993) from the first stage of spermatogenesis, and temporary high expression of the apoptosis-promoting its perturbation may extend to the life-long process of protein Bax, which disappears at sexual maturity. mature spermatogenesis. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal Results mouse testis) in this early apoptotic wave is shown byMale transgenic mice expressing Bcl2 or the occurrence of a comparable testicular syndrome in overexpressing BclxL in their germinal cells are mice defective in the bax gene. The apoptotic wave sterile and display marked alterations in the late appears necessary for normal mature spermatogenesis stages of spermatogenesis to develop, probably because it maintains a critical cellMice bearing a human bcl2 or a human bclx transgene number ratio between some germinal cell stages and placed under the control of the promoter of the murine Sertoli cells, whose normal functions and differentihousekeeping phosphoglycerate kinase-1 (pgk-1) gene ation involve an elaborate network of communication.were generated. One male and four female founders Keywords: apoptosis/germinal cells/spermatogenesis/ bearing a bcl2 transgene were obtained. The male (T41) transgenic was sterile and one of the females (T56) showed a lack of vaginal opening at sexual maturity. The ovaries of this founder female were, however, functional, since, when grafted onto ovariectomized normal female mice, they

SummaryBreaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic.

SummaryMice injected with anti-Fas antibody die within a few hours with total liver destruction due to massive apoptosis of hepatocytes. We show that this is preceded and accompanied by the sequential activation of cysteine proteases of the interleukin 1[3-converting enzyme (ICE) and CPP32 types in the cytosol of the hepatocytes, and that proCPP32 cleavage and enzymatic activity can be prevented by intravenous injections of the tripeptide N-benzyloxycarbonyl-Val-AlaAsp-fluoromethylketone (Z-VAD.fmk), an inhibitor of ICE-like proteases. Four Z-VAD.fmk injections at 1-hour intervals abolished all signs of liver damage after anti-Fas antibody injection and resulted in 100% long-range recovery, without residual tissue damage, from a condition otherwise uniformly fatal within <3 hours. This treatment was effective even when delayed unnl some liver DNA degradation was already detectable. Injections of the tetrapeptide Ac-YVAD.cmk, more specific for the ICE-like subfamily of cysteine proteases but less cell permeable, also gave protection, but at higher doses and when injections started before that of anti-Fas antibody. These observations afford a way of temporarily modulating a number of apoptotic processes in vivo and may have important therapeutic implications in some human diseases. There is now conclusive evidence that the process of apoptosis or programmed cell death (PCD) results from the activation of members of a new family of cysteine proteases with a specificity of cleavage for aspartate in the P1 position. The decisive importance of this mechanism as an effector of PCD was revealed by the discovery that the ced-3 gene of Caenorhabditis elegans, which is required for cell death occurring during the normal development of this nematode (1), encodes for a protein related to the mammalian IL-l[3-converting enzyme (ICE) (2), an aspartate-specific cysteine protease, and that, in certain conditions, overexpression of ICE itself in mammalian cells can lead to apoptosis (3). Several other members of this protease family have now been identified and are presently subdivided into three subfamilies, each containing variants: the ICE, CPP32 (also called YAMA, apopain, or priCE), and Ich-1 (or Nedd-2) subfamilies; all these enzymes are synthesized as inactive proenzymes requiring cleavage at specific Asp residues to be transformed into active proteases: thus, at least some of these proteases can activate each other in the form of a proteolytic cascade and/or may undergo, once activated, autoprocessing, allowing the potentially lethal amplification of a minor imtial proteolytic process (for review, see reference 4). Short peptides corresponding to the cleavage site of some of these cysteine proteases have been used as inhibitors, with the Tyr-Val-Ala-Asp (YVAD) and the Asp-Glu-Val-Asp (DEVD) motifs being rather specific inhibitors of the ICE and CPP32 subfamilies, respectively (5). Provided their extracellular concentration is high enough, addition of these or related inhibitory peptides to cell cultures prevents many form...

IntroductionTargeting leukocyte migration from the vasculature to sites of inflammation requires a series of coordinated adhesive interactions. 1 Of particular interest are molecules distributed at endothelial junctions, where cis-and trans-interactions allow endothelial cells (ECs) to interact with and regulate leukocyte migration into underlying tissues.Junctional adhesion molecules (JAMs) encompass a family of 6 immunoglobulin-like proteins: CAR, ESAM, JAM4, JAM-A, JAM-B, and JAM-C. 2,3 Differential expression and redistribution of JAM-B and JAM-C at the endothelial junction contribute to leukocyte interactions and trafficking. 4 Endothelial cell JAM-C preferentially interacts with JAM-B, forming a 2-dimensional network of both molecules localized at endothelial junctions. [4][5][6] Complex hierarchies dictate how JAM-B/-C multimers interact with integrin counterreceptors. JAM-B can bind the integrin VLA-4 (␣ 4 ␤ 1 ; CD29a/CD49d), but requires prior engagement with JAM-C, 7 whereas JAM-C can act as a counterreceptor for the integrin Mac-1 (␣ M ␤ 2 ; CD11b/CD18) independently of Although JAM-C is expressed by fibroblasts 9 and epithelial cells, 10-12 the role of endothelial JAM-C in leukocyte accumulation during inflammation 13 has received particular attention. In humans, JAM-C is expressed on circulating platelets, natural killer (NK) cells, dendritic cells, and subsets of T and B cells, 4,7,14-16 but not on circulating mouse leukocytes. 5 Preliminary studies with functional blocking antibodies to JAM-C showed reduced transmigration of peripheral blood lymphocytes across cultured human umbilical vein endothelial cells (HUVECs), 15 and later studies identified Mac-1 as a ligand partner that can mediate adhesion and transmigration for neutrophils and monocytes and transepithelial migration of neutrophils. 8,10,17,18 Numerous studies have addressed this dual functionality of JAM-C as an adhesion and transmigration regulatory molecule. Experimental animal models of inflammation support a role for JAM-C in regulating leukocyte accumulation within inflammatory lesions. Soluble JAM-C (solJAM-C) and antibodies to JAM-C inhibit leukocyte emigration in mouse models of inflammation. 5,17 In addition, in mouse models of allergic contact dermatitis, the regulatory role of JAM-C in inflammation has also been associated with modulation of JAM-B/-C interactions, where combined blocking antibodies to JAM-B and JAM-C had an additive effect on blocking leukocyte recruitment. 19 Furthermore, recent reports have identified additional steps requiring the disruption/engagement of the JAM-B/-C complex, before the respective integrin counterreceptor is engaged. 6,7 In this study, we have developed a flow assay that allows detailed analysis of individual monocyte interactions with ECs. This analysis can begin at the initial phase of capture from free-flow under physiologic conditions and continue to posttransmigrational events. Using this model, we elucidated a novel role for endothelial JAM-C in regulating monocyte retention in t...

Targeted disruption of mouse ␤ 3 -adrenoceptor was generated by homologous recombination, and validated by an acute in vivo study showing a complete lack of effect of the ␤ 3 -adrenoceptor agonist CL 316,243 on the metabolic rate of homozygous null ( Ϫ / Ϫ ) mice. In brown adipose tissue, ␤ 3 -adrenoceptor disruption induced a 66% decrease ( P Ͻ 0.005) in ␤ 1 -adrenoceptor mRNA level, whereas leptin mRNA remained unchanged. Chronic energy balance studies in chow-fed mice showed that in Ϫ / Ϫ mice, body fat accumulation was favored ( ϩ 41%, P Ͻ 0.01), with a slight increase in food intake ( ϩ 6%, NS). These effects were accentuated by high fat feeding: Ϫ / Ϫ mice showed increased total body fat ( ϩ 56%, P Ͻ 0.025) and food intake ( ϩ 12%, P Ͻ 0.01), and a decrease in the fat-free dry mass ( Ϫ 10%, P Ͻ 0.05), which reflects a reduction in body protein content. Circulating leptin levels were not different in Ϫ / Ϫ and control mice regardless of diet. The significant shift to the right in the positive correlation between circulating leptin and percentage of body fat in high fat-fed Ϫ / Ϫ mice suggests that the threshold of body fat content inducing leptin secretion is higher in Ϫ / Ϫ than in control mice. Taken together, these studies demonstrate that ␤ 3 -adrenoceptor disruption creates conditions which predispose to the development of obesity. ( J. Clin. Invest. 1997. 100:1098-1106.)

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Junctional adhesion molecule C (JAM-C) is expressed by vascular endothelium and human but not mouse B lymphocytes.

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