Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair.

After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report generation of an anti-ceramide 6B5 single-chain variable fragment (scFv) and show that s.c. 6B5 scFv delivery at 24 hours after a 90% lethal GI-ARS dose of 15 Gy mitigated mouse lethality, despite administration after DNA repair was complete. We defined an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis after radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ, and mouse survival. We posit that successful preclinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.

Fusion of lysosomes to plasma membrane initiates radiation-induced apoptosis.

Diverse stresses, including reactive oxygen species (ROS), ionizing radiation, and chemotherapies, activate acid sphingomyelinase (ASMase) and generate the second messenger ceramide at plasma membranes, triggering apoptosis in specific cells, such as hematopoietic cells and endothelium. Ceramide elevation drives local bilayer reorganization into ceramide-rich platforms, macrodomains (0.5-5-µm diameter) that transmit apoptotic signals. An unresolved issue is how ASMase residing within lysosomes is released extracellularly within seconds to hydrolyze sphingomyelin preferentially enriched in outer plasma membranes. Here we show that physical damage by ionizing radiation and ROS induces full-thickness membrane disruption that allows local calcium influx, membrane lysosome fusion, and ASMase release. Further, electron microscopy reveals that plasma membrane "nanopore-like" structures (∼100-nm diameter) form rapidly due to lipid peroxidation, allowing calcium entry to initiate lysosome fusion. We posit that the extent of upstream damage to mammalian plasma membranes, calibrated by severity of nanopore-mediated local calcium influx for lysosome fusion, represents a biophysical mechanism for cell death induction.

Organoids Reveal That Inherent Radiosensitivity of Small and Large Intestinal Stem Cells Determines Organ Sensitivity.

Tissue survival responses to ionizing radiation are nonlinear with dose, rather yielding tissue-specific descending curves that impede straightforward analysis of biologic effects. Apoptotic cell death often occurs at low doses, while at clinically relevant intermediate doses, double-strand break misrepair yields mitotic death that determines outcome. As researchers frequently use a single low dose for experimentation, such strategies may inaccurately depict inherent tissue responses. Cutting edge radiobiology has adopted full dose survival profiling and devised mathematical algorithms to fit curves to observed data to generate highly reproducible numerical data that accurately define clinically relevant inherent radiosensitivities. Here, we established a protocol for irradiating organoids that delivers radiation profiles simulating the organ of origin. This technique yielded highly similar dose-survival curves of small and large intestinal crypts in vivo and their cognate organoids analyzed by the single-hit multi-target (SHMT) algorithm, outcomes reflecting the inherent radiation profile of their respective Lgr5+ stem cell populations. As this technological advance is quantitative, it will be useful for accurate evaluation of intestinal (patho)physiology and drug screening. SIGNIFICANCE: These findings establish standards for irradiating organoids that deliver radiation profiles that phenocopy the organ of origin.See related commentary by Muschel et al., p. 927.

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent.

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of ≤0.25 µg/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 µg/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

Targeting acid sphingomyelinase with anti-angiogenic chemotherapy.

Despite great promise, combining anti-angiogenic and conventional anti-cancer drugs has produced limited therapeutic benefit in clinical trials, presumably because mechanisms of anti-angiogenic tissue response remain only partially understood. Here we define a new paradigm, in which anti-angiogenic drugs can be used to chemosensitize tumors by targeting the endothelial acid sphingomyelinase (ASMase) signal transduction pathway. We demonstrate that paclitaxel and etoposide, but not cisplatin, confer ASMase-mediated endothelial injury within minutes. This rapid reaction is required for human HCT-116 colon cancer xenograft complete response and growth delay. Whereas VEGF inhibits ASMase, anti-VEGFR2 antibodies de-repress ASMase, enhancing endothelial apoptosis and drug-induced tumor response in asmase+/+, but not in asmase-/-, hosts. Such chemosensitization occurs only if the anti-angiogenic drug is delivered 1-2h before chemotherapy, but at no other time prior to or post chemotherapy. Our studies suggest that precisely-timed administration of anti-angiogenic drugs in combination with ASMase-targeting anti-cancer drugs is likely to optimize anti-tumor effects of systemic chemotherapy. This strategy warrants evaluation in future clinical trials.

Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic alone for treating methicillin-resistant Staphylococcus aureus-induced murine bacteremia.

Lysins are bacteriophage-derived enzymes that degrade bacterial peptidoglycans. Lysin CF-301 is being developed to treat Staphylococcus aureus because of its potent, specific, and rapid bacteriolytic effects. It also demonstrates activity on drug-resistant strains, has a low resistance profile, eradicates biofilms, and acts synergistically with antibiotics. CF-301 was bacteriolytic against 250 S. aureus strains tested including 120 methicillin-resistant S. aureus (MRSA) isolates. In time-kill studies with 62 strains, CF-301 reduced S. aureus by 3-log10 within 30 minutes compared to 6-12 hours required by antibiotics. In bacteremia, CF-301 increased survival by reducing blood MRSA 100-fold within 1 hour. Combinations of CF-301 with vancomycin or daptomycin synergized in vitro and increased survival significantly in staphylococcal-induced bacteremia compared to treatment with antibiotics alone (P < .0001). Superiority of CF-301 combinations with antibiotics was confirmed in 26 independent bacteremia studies. Combinations including CF-301 and antibiotics represent an attractive alternative to antibiotic monotherapies currently used to treat S. aureus bacteremia.

Adenoviral transduction of human acid sphingomyelinase into neo-angiogenic endothelium radiosensitizes tumor cure.

These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, PPE1(3x), and a hypoxia-inducible dual-binding HIF-2α-Ets-1 enhancer element upstream of the asmase gene, inserted into a replication-deficient adenovirus yielding the vector Ad5H2E-PPE1(3x)-ASMase. This vector confers ASMase over-expression in cycling angiogenic endothelium in vitro and within tumors in vivo, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with Ad5H2E-PPE1(3x)-ASMase markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, Ad5H2E-PPE1(3x)-ASMase treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors.

Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice.

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.

Antitumor efficacy following the intracellular and interstitial release of liposomal doxorubicin.

pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.

Accelerated hematopoietic toxicity by high energy (56)Fe radiation.

PURPOSE: There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or X-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. METHODS: C57BL/6J mice were irradiated with (56)Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of (56)Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. RESULTS: Although onset was more rapid, (56)Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)(50/30) (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy, respectively, with relative biologic effectiveness for (56)Fe ions of 1.25 and 1.06 for protons. CONCLUSIONS: (56)Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity.

Ceacam1 separates graft-versus-host-disease from graft-versus-tumor activity after experimental allogeneic bone marrow transplantation.

BACKGROUND: Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models. METHODS: We studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1(-/-) T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25(hi), CD62L(lo)). Additionally, Ceacam1(-/-) CD8 T cells had greater expression of the gut-trafficking integrin α(4)ß(7), though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1(-/-) recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1(-/-) mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1(+) lymphoma model was improved in animals receiving Ceacam1(-/-) vs. control T cells. CONCLUSIONS: We conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.

Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

BACKGROUND: Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates. METHODOLOGY/PRINCIPAL FINDINGS: MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax. CONCLUSIONS/SIGNIFICANCE: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

Alpha particles induce apoptosis through the sphingomyelin pathway.

The sphingomyelin pathway involves the enzymatic cleavage of sphingomyelin to produce ceramide, a second messenger that serves as a key mediator in the rapid apoptotic response to various cell stressors. Low-linear energy transfer (LET) γ radiation can initiate this pathway, independent of DNA damage, via the cell membrane. Whether short-ranged, high-LET α particles, which are of interest as potent environmental carcinogens, radiotherapies and potential components of dirty bombs, can act through this mechanism to signal apoptosis is unknown. Here we show that irradiation of Jurkat cells with α particles emitted by the ²²5Ac-DOTA-anti-CD3 IgG antibody construct results in dose-dependent apoptosis. This apoptosis was significantly reduced by pretreating cells with cholesterol-depleting nystatin, a reagent known to inhibit ceramide signaling by interfering with membrane raft coalescence and ceramide-rich platform generation. The effects of nystatin on α-particle-induced apoptosis were related to disruption of the ceramide pathway and not to microdosimetry alterations, because similar results were obtained after external irradiation of the cells with a broad beam of collimated α particles using a planar ²4¹Am source. External irradiation allowed for more precise control of the dosimetry and geometry of the irradiation, independent of antibody binding or cell internalization kinetics. Mechanistically consistent with these findings, Jurkat cells rapidly increased membrane concentrations of ceramide after external irradiation with an average of five α-particle traversals per cell. These data indicate that α particles can activate the sphingomyelin pathway to induce apoptosis.

Regulation of ceramide synthase-mediated crypt epithelium apoptosis by DNA damage repair enzymes.

Acute endothelial cell apoptosis and microvascular compromise couple gastrointestinal tract irradiation to reproductive death of intestinal crypt stem cell clonogens (SCCs) following high-dose radiation. Genetic or pharmacologic inhibition of endothelial apoptosis prevents intestinal damage, but as the radiation dose is escalated, SCCs become directly susceptible to an alternate cell death mechanism, mediated via ceramide synthase (CS)-stimulated de novo synthesis of the proapoptotic sphingolipid ceramide, and p53-independent apoptosis of crypt SCCs. We previously reported that ataxia-telangiectasia mutated deficiency resets the primary radiation lethal pathway, allowing CS-mediated apoptosis at the low-dose range of radiation. The mechanism for this event, termed target reordering, remains unknown. Here, we show that inactivation of DNA damage repair pathways signals CS-mediated apoptosis in crypt SCCs, presumably via persistent unrepaired DNA double-strand breaks (DSBs). Genetic loss of function of sensors and transducers of DNA DSB repair confers the CS-mediated lethal pathway in intestines of sv129/B6Mre11(ATLD1/ATLD1) and C57BL/6(Prkdc/SCID) (severe combined immunodeficient) mice exposed to low-dose radiation. In contrast, CS-mediated SCC lethality was mitigated in irradiated gain-of-function Rad50(s/s) mice, and epistasis studies order Rad50 upstream of Mre11. These studies suggest unrepaired DNA DSBs as causative in target reordering in intestinal SCCs. As such, we provide an in vivo model of DNA damage repair that is standardized, can be exploited to understand allele-specific regulation in intact tissue, and is pharmacologically tractable.

The use of pH-triggered leaky heterogeneities on rigid lipid bilayers to improve intracellular trafficking and therapeutic potential of targeted liposomal immunochemotherapy.

During endocytosis, pH-triggered release of encapsulated therapeutics from delivery carriers may accelerate their intracellular trafficking increasing therapeutic efficacy. To improve the therapeutic potential of targeted immunochemotherapy using anti-HER2/neu liposomal doxorubicin, we exploit the formation of leaky heterogeneities on rigid lipid bilayers to extensively release doxorubicin during endocytosis. We have previously demonstrated that pH-dependent formation of phase-separated lipid heterogeneities on the plane of a bilayer membrane increases the permeability of bilayers when they are composed of lipid pairs with rigid non-matching acyl chain lengths. This was suggested to be due to defective packing among lipids residing at the interfaces of lipid domains. Here we design nanometer-size antiHER2/neu-labeled PEGylated vesicles composed of lipid pairs with longer non-matching acyl chain lengths (n=18 and 21). These vesicles exhibit superior killing efficacy of cancer cells compared to established liposome formulations, and their killing efficacy is similar to the effect of combined free doxorubicin and free antiHER2/neu antibody. Other transport-related properties such as liposome blood circulation times, and specific binding and internalization by cancer cells are unaffected. These results demonstrate the potential of vesicles with pH-triggered leaky heterogeneities to increase the therapeutic potential of targeted immunochemotherapy.

Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease.

Alloreactive donor cytolytic T lymphocytes play a critical role in pathophysiology of acute graft-versus-host disease (GVHD). As GVHD progression involves tumor necrosis factor superfamily receptor activation, and as apoptotic signaling for some tumor necrosis factor superfamily receptors might involve acid sphingomyelinase (ASMase)-mediated ceramide generation, we hypothesized that ASMase deletion would ameliorate GVHD. Using clinically relevant mouse models of acute GVHD in which allogeneic bone marrow and T cells were transplanted into asmase+/+ and asmase(-/-) hosts, we identify host ASMase as critical for full-blown GVHD. Lack of host ASMase reduced the acute inflammatory phase of GVHD, attenuating cytokine storm, CD8+ T-cell proliferation/activation, and apoptosis of relevant graft-versus-host target cells (hepatocytes, intestinal, and skin cells). Organ injury was diminished in asmase(-/-) hosts, and morbidity and mortality improved at 90 days after transplantation. Resistance to cytolytic T lymphocyte-induced apoptosis was found at the target cell membrane if hepatocytes lack ASMase, as hepatocyte apoptosis required target cell ceramide generation for formation of ceramide-rich macrodomains, sites concentrating proapoptotic Fas. These studies indicate a requirement for target cell ASMase in evolution of GVHD in liver, small intestines, and skin and provide potential new targets for disease management.