Retraction Note: Microglia-dependent synapse loss in type I interferon-mediated lupus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microglia-dependent synapse loss in type I interferon-mediated lupus.

Systemic lupus erythematosus (SLE) is an incurable autoimmune disease characterized by autoantibody deposition in tissues such as kidney, skin and lungs. Notably, up to 75% of patients with SLE experience neuropsychiatric symptoms that range from anxiety, depression and cognitive impairment to seizures and, in rare cases, psychosis-collectively this is referred to as central nervous system (CNS) lupus. In some cases, certain autoantibodies, such as anti-NMDAR or anti-phospholipid antibodies, promote CNS lupus. However, in most patients, the mechanisms that underlie these symptoms are unknown. CNS lupus typically presents at lupus diagnosis or within the first year, suggesting that early factors contributing to peripheral autoimmunity may promote CNS lupus symptoms. Here we report behavioural phenotypes and synapse loss in lupus-prone mice that are prevented by blocking type I interferon (IFN) signalling. Furthermore, we show that type I IFN stimulates microglia to become reactive and engulf neuronal and synaptic material in lupus-prone mice. These findings and our observation of increased type I IFN signalling in post-mortem hippocampal brain sections from patients with SLE may instruct the evaluation of ongoing clinical trials of anifrolumab, a type I IFN-receptor antagonist. Moreover, identification of IFN-driven microglia-dependent synapse loss, along with microglia transcriptome data, connects CNS lupus with other CNS diseases and provides an explanation for the neurological symptoms observed in some patients with SLE.

Intracellular Activation of Complement 3 Is Responsible for Intestinal Tissue Damage during Mesenteric Ischemia.

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B-deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.

Platelets orchestrate remote tissue damage after mesenteric ischemia-reperfusion.

Ischemia-reperfusion (I/R) injury is a leading cause of morbidity and mortality. A functional role for platelets in tissue damage after mesenteric I/R is largely unknown. The hypothesis that mesenteric I/R local and remote injury are platelet dependent was tested. Using a murine mesenteric I/R model, we demonstrate that platelets orchestrate remote lung tissue damage that follows mesenteric I/R injury and also contribute, albeit to a lesser degree, to local villi damage. While lung damage is delayed compared with villi damage, it increased over time and was characterized by accumulation of platelets in the pulmonary vasculature early, followed by alveolar capillaries and extravasation into the pulmonary space. Both villi and lung tissues displayed complement deposition. We demonstrate that villi and lung damage are reduced in mice made platelet deficient before I/R injury and that platelet transfusion into previously platelet-depleted mice before I/R increased both villi and lung tissue damage. Increased C3 deposition accompanied platelet sequestration in the lung, which was mostly absent in platelet-depleted mice. In contrast, C3 deposition was only minimally reduced on villi of platelet-depleted mice. Our findings position platelets alongside complement as a significant early upstream component that orchestrates remote lung tissue damage after mesenteric I/R and strongly suggest that reperfusion injury mitigating modalities should consider the contribution of platelets.

Inhibition of Syk activity by R788 in platelets prevents remote lung tissue damage after mesenteric ischemia-reperfusion injury.

Tissue injury following ischemia-reperfusion (I/R) occurs as a consequence of actions of soluble factors and immune cells. Growing evidence supports a role for platelets in the manifestation of tissue damage following I/R. Spleen tyrosine kinase has been well documented to be important in lymphocyte activation and more recently in platelet activation. We performed experiments to evaluate whether inhibition of platelet activation through inhibition of spleen tyrosine kinase prevents tissue damage after mesenteric I/R injury. Platelets isolated from C57BL/6J mice fed with R788 for 10 days were transfused into C57BL/6J mice depleted of platelets 2 days before mesenteric I/R injury. Platelet-depleted mice transfused with platelets from R788-treated mice before mesenteric I/R displayed a significant reduction in the degree of remote lung damage, but with little change in the degree of local intestinal damage compared with control I/R mice. Transfusion of R788-treated platelets also decreased platelet sequestration, C3 deposition, and immunoglobulin deposition in lung, but not in the intestine, compared with control groups. These findings demonstrate that platelet activation is a requisite for sequestration in the pulmonary vasculature to mediate remote tissue injury after mesenteric I/R. The use of small-molecule inhibitors may be valuable to prevent tissue damage in remote organs following I/R injury.

Depletion of gut commensal bacteria attenuates intestinal ischemia/reperfusion injury.

Gut commensal bacteria play important roles in the development and homeostasis of intestinal immunity. However, the role of gut commensals in intestinal ischemia/reperfusion (I/R) injury is unclear. To determine the roles of gut commensal bacteria in intestinal IR injury, we depleted gut microbiota with a broad-spectrum antibiotic cocktail and performed mesenteric I/R (M I/R). First, we confirmed that antibiotic treatment completely depleted gut commensal bacteria and diminished the size of secondary lymphoid tissues such as the Peyer's patches. We next found that antibiotic treatment attenuated intestinal injury following M I/R. Depletion of gut commensal bacteria reduced the expression of Toll-like receptor (TLR)2 and TLR4 in the intestine. Both are well-known receptors for gram-positive and -negative bacteria. Decreased expression of TLR2 and TLR4 led to the reduction of inflammatory mediators, such as TNF, IL-6, and cyclooxygenase-2. Intestinal I/R injury is initiated when natural antibodies recognize neo-antigens that are revealed on ischemic cells and activate the complement pathway. Thus we evaluated complement and immunoglobulin (Ig) deposition in the damaged intestine and found that antibiotic treatment decreased the deposition of both C3 and IgM. Interestingly, we also found that the deposition of IgA also increased in the intestine following M I/R compared with control mice and that antibiotic treatment decreased the deposition of IgA in the damaged intestine. These results suggest that depletion of gut commensal bacteria decreases B cells, Igs, and TLR expression in the intestine, inhibits complement activation, and attenuates intestinal inflammation and injury following M I/R.

Spleen tyrosine kinase inhibition prevents tissue damage after ischemia-reperfusion.

Reperfusion injury to tissue following an ischemic event occurs as a consequence of an acute inflammatory response that can cause significant morbidity and mortality. Components of both the innate (complement, immunoglobulin, monocytes, and neutrophils) and adaptive (B and T lymphocytes) immune systems have been demonstrated to mediate tissue injury. Spleen tyrosine kinase (Syk) is responsible for membrane-mediated signaling in various cell types including B lymphocytes, macrophages, and T cells. We investigated the ability of a small drug Syk inhibitor, R788, to protect mice against mesenteric ischemia-reperfusion (I/R)-induced local (intestine) and remote lung injury. Mice were fed with chow containing a Syk inhibitor for 6 days before the performance of intestinal I/R, which resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local and remote lung injury. The beneficial effect was associated with reduced IgM and complement 3 deposition in the tissues and significant reduction of polymorphonuclear cell infiltration. Our data place Syk upstream of events leading to the binding of natural antibodies to the ischemia-conditioned tissues and urge the consideration of the use of Syk inhibitors in the prevention or improvement of tissue injury of organs exposed to ischemia or hypoperfusion.

Complement Deposition on the Surface of RBC After Trauma Serves a Biomarker of Moderate Trauma Severity: A Prospective Study.

BACKGROUND: Activation of the complement system and complement deposition on red blood cells (RBCs) contribute to organ damage in trauma. We conducted a prospective study in subjects with traumatic injuries to determine the pattern of complement deposition on RBC and whether they are associated with clinical outcomes. METHOD: A total of 124 trauma patients and 42 healthy controls were enrolled in this prospective study. RBC and sera were collected at 0, 6, 24, and 72 h from trauma patients and healthy controls during a single draw. Presence of C4d, C3d, C5b-9, phosphorylation of band 3 and production of nitric oxide were analyzed by flow cytometry. RESULTS: RBC from trauma patients at all time points up to 24 h displayed significantly higher deposition of C4d on their RBC membrane as compared with healthy donors. Incubation of normal RBC with sera from trauma patients resulted in significant increase of C4d deposition (at 0, 6, 24, and 72 h), C5b-9 deposition (at 0 and 6 h), phosphorylation of band 3 (at 0 and 24 h), and nitric oxide production up to 24 h compared with sera from healthy subjects. Deposition of C4d and C5b-9 in patients with an Injury Severity Score (ISS) of 9 and above remained elevated up to 72 h. CONCLUSIONS: Our study demonstrates that the presence of C4d, C3d, and C5b-9 on the surface of RBC is linked to increased phosphorylation of band 3 and increased production of nitric oxide. Deposition of C4d and C5b-9 decreased faster over course of 3-day study in subjects with ISS less than 9.

Complement and coagulation cascades in trauma.

Trauma remains a major cause of death throughout the world, especially for patients younger than 45 years. Due to rapid advances in clinical management, both in the acute and prehospital settings, trauma patients survive devastating injuries at unprecedented rates. However, these patients can often face life threatening complications that stem from the robust innate immune response induced by severe hemorrhage, leading to further tissue injury rather than repair. The complement and coagulation cascades are key mediators in this disordered reaction, which includes the development of trauma-induced coagulopathy. There is increasing evidence that cross-talk between these two pathways allows rapid amplification of their otherwise targeted responses and contributes to overwhelming and prolonged systemic inflammation. In this article, we summarize the initial steps of innate immune response to trauma and review the complex complement and coagulation cascades, as well as how they interact with each other. Despite progress in understanding these cascades, effective therapeutic targets have yet to be found and further research is needed both to improve survival rates as well as decrease associated morbidity.

Hyaluronic Acid Synthesis Contributes to Tissue Damage in Systemic Lupus Erythematosus.

Hyaluronic acid (HA), a component of the extracellular matrix, is the ligand for CD44 and has been implicated in the pathogenesis of kidney inflammation in patients with systemic lupus erythematosus (SLE), but its direct role and mechanism of action have not been studied. Here we show that administration of hymecromone (4-Methylumbelliferone, 4-MU), an HA synthesis inhibitor, to lupus-prone mice suppressed dramatically lupus-related pathology. Interestingly, 4-MU stopped the appearance of disease when administered prior to its onset and inhibited the progression of disease when administered after its appearance. Inhibition of HA synthesis in vivo reduced tissue damage and the number of intrarenal lymphoid cell infiltrates including double negative CD3+CD4-CD8- T cells which are known to be involved in the pathogenesis of SLE. Exposure of human peripheral blood mononuclear cells to HA in vitro increased the generation of CD3+CD4-CD8- T cells through a mechanism involving Rho-associated kinase. Our results signify the importance of the HA-rich tissue microenvironment in the activation of lymphocytes to cause tissue damage in SLE and suggest the consideration of inhibition of HA synthesis to treat patients.

C3a Enhances the Formation of Intestinal Organoids through C3aR1.

C3a is important in the regulation of the immune response as well as in the development of organ inflammation and injury. Furthermore, C3a contributes to liver regeneration but its role in intestinal stem cell function has not been studied. We hypothesized that C3a is important for intestinal repair and regeneration. Intestinal organoid formation, a measure of stem cell capacity, was significantly limited in C3-deficient and C3a receptor (C3aR) 1-deficient mice while C3a promoted the growth of organoids from normal mice by supporting Wnt-signaling but not from C3aR1-deficient mice. Similarly, the presence of C3a in media enhanced the expression of the intestinal stem cell marker leucine-rich repeat G-protein-coupled receptor 5 (Lgr5) and of the cell proliferation marker Ki67 in organoids formed from C3-deficient but not from C3aR1-deficient mice. Using Lgr5.egfp mice we showed significant expression of C3 in Lgr5+ intestinal stem cells whereas C3aR1 was expressed on the surface of various intestinal cells. C3 and C3aR1 expression was induced in intestinal crypts in response to ischemia/reperfusion injury. Finally, C3aR1-deficient mice displayed ischemia/reperfusion injury comparable to control mice. These data suggest that C3a through interaction with C3aR1 enhances stem cell expansion and organoid formation and as such may have a role in intestinal regeneration.

Platelet-associated CD40/CD154 mediates remote tissue damage after mesenteric ischemia/reperfusion injury.

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage.

The role of platelet factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury.

The robust inflammatory response that occurs during ischemia reperfusion (IR) injury recruits factors from both the innate and adaptive immune systems. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR injury has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration were decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissues. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after IR at both the mRNA and protein levels. In conclusion, these findings demonstrate that may PF4 represent an important mediator of local and remote tissue damage.

Ischemia-mediated aggregation of the actin cytoskeleton is one of the major initial events resulting in ischemia-reperfusion injury.

Ischemia-reperfusion (IR) injury represents a major clinical challenge, which contributes to morbidity and mortality during surgery. The critical role of natural immunoglobulin M (IgM) and complement in tissue injury has been demonstrated. However, cellular mechanisms that result in the deposition of natural IgM and the activation of complement are still unclear. In this report, using a murine intestinal IR injury model, we demonstrated that the beta-actin protein in the small intestine was cleaved and actin filaments in the columnar epithelial cells were aggregated after a transient disruption during 30 min of ischemia. Ischemia also led to deposition of natural IgM and complement 3 (C3). A low dose of cytochalasin D, a depolymerization reagent of the actin cytoskeleton, attenuated this deposition and also attenuated intestinal tissue injury in a dose-dependent manner. In contrast, high doses of cytochalasin D failed to worsen the injury. These data indicate that ischemia-mediated aggregation of the actin cytoskeleton, rather than its disruption, results directly in the deposition of natural IgM and C3. We conclude that ischemia-mediated aggregation of the actin cytoskeleton leads to the deposition of natural IgM and the activation of complement, as well as tissue injury.

Murine dendritic cell antigen-presenting cell function is not altered by burn injury.

Severe injury disrupts normal immune regulation causing a transient hyperinflammatory reaction and suppressed adaptive immune function. This report addresses the potential contribution of dendritic cells (DC) to changes in adaptive immune function after injury by specifically measuring injury-induced changes in splenic DC numbers and subsets, cell-surface markers, TLR responses, and APC function. Using a mouse burn injury model, we found that injury did not markedly alter the relative percentage of lymphoid, myeloid, or plasmacytoid DC in the spleens of burn-injured mice. Moreover, we did not observe a significant reduction in cell-surface expression of several major costimulatory molecules, CD40, CD80, CD86, programmed death 1 ligand, ICOS ligand, and B7-H3, on DC. Instead, we observed increased cell-surface expression of CD86 at 1 day after injury with no significant changes in costimulatory molecule expression at 7 days after injury, suggesting that burn injury causes an early activation of DC. In addition, injury did not suppress DC reactivity to TLR2, TLR4, or TLR9 agonists. Most important, DC prepared from injured mice were able to present peptide antigen to naive OTII TCR transgenic CD4+ T cells as efficiently and effectively as DC from sham-injured mice. We also found that CD4 T cells stimulated with antigen presented by DC from sham or burn mice showed similar levels of IL-2, IFN-gamma, IL-10, and IL-13 production. Taken together, these findings support the conclusion that DC do not acquire a suppressive phenotype following severe injury in mice.

Platelet depletion in mice increases mortality after thermal injury.

Platelets play a fundamental role in maintaining hemostasis and have been shown to participate in innate and adaptive immunity. However, the role of platelets in the immune response to injury remains undefined. We tested the importance of platelets in the host response to serious injury in a newly developed platelet-deficient mouse model. Wild-type and platelet-depleted C57BL/6J mice underwent a 25% full-thickness total body surface area thermal or sham injury. Platelet-deficient mice showed survival of 51% at 48 hours after injury compared with 94% to 100% survival in experimental control mice (P < .001). Necropsy and histology ruled out hemorrhage and hypovolemia as causes of death. Percentages of peripheral blood monocytes (P < .01) and neutrophils (P < .05) were increased between 36 and 48 hours after thermal injury in platelet-deficient mice compared with control mice. Plasma levels of TNFalpha (P < .001), IL-6 (P < .001), and MCP-1 (P < .05) were also elevated by 24 hours whereas levels of TGFbeta(1) were reduced between 24 and 36 hours following injury in platelet-depleted mice (P < .001) compared with control mice. Our findings demonstrate for the first time that platelets play a critical protective role during the host response to injury. Moreover, our findings suggest that platelets and, more importantly, platelet-derived TGFbeta(1) modulate the systemic inflammatory response occurring after injury.

Increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity.

OBJECTIVES: We recently reported increased CD4 CD25 T regulatory (Treg) activity after burn injury in mice. This study sought to determine if Tregs mediate the reduction in TH1-type immunity after serious injury in man and if Treg function is altered by injury. METHODS: Peripheral blood was withdrawn from 19 consenting adult patients (35.1 +/- 16.3 years of age) with Injury Severity Scores (ISS) 36.6 +/- 13.9 on days 1 and 7 after trauma and from 5 healthy individuals. CD4 T cells were purified and sorted into Treg (CD25(high)) and Treg-depleted populations. After activation of cells with anti-CD3/CD28 antibody, production of the TH1-type cytokine IFNgamma, TH2-type cytokines (IL-4 and IL-5), and the inhibitory cytokine IL-10 was measured using cytometric bead arrays. Treg activity was measured by in vitro suppression of autologous CD4 T cell proliferation. RESULTS: All patients survived, 9 (47%) developed infection postinjury. IFNgamma production by patient CD4 T cells was decreased on day 1 and day 7, when compared with healthy controls. However, when Tregs were depleted from the CD4 T cells, the IFNgamma production increased to control levels. Tregs were the chief source of IL-4 and IL-5 as well as IL-10. Treg suppression of T cell proliferation increased significantly from day 1 to day 7 after injury. CONCLUSIONS: We demonstrate for the first time that human Tregs are increased in potency after severe injury. Most significantly, Tregs are important mediators of the suppression of T cell activation and the reduction in TH1 cytokine production found after injury.

The CD40-induced signaling pathway in endothelial cells resulting in the overexpression of vascular endothelial growth factor involves Ras and phosphatidylinositol 3-kinase.

Ligation of endothelial cell (EC) CD40 induces the expression of several proinflammatory cytokines as well as angiogenesis factors, including vascular endothelial growth factor (VEGF). Moreover, despite the reported importance of CD40 in cell-mediated immunity, little is known of the CD40-induced signaling pathways in EC. In this study, we have investigated the function of the Ras signaling pathway(s) for CD40-induced overexpression of VEGF. EC were transiently transfected with a full-length VEGF promoter-luciferase construct and a dominant-inhibitory mutant of Ras (Ras17N). Following transfection, ligation of CD40 with soluble CD40 ligand resulted in a significant increase in VEGF transcriptional activation, and the inhibitory mutant of Ras blocked this CD40-induced VEGF overexpression. Using EMSA and Western blot analysis, we demonstrated that CD40-dependent binding of nuclear protein(s) to the VEGF promoter and CD40-induced VEGF protein expression in EC were also inhibited by the Ras mutant. Immunoprecipitation studies revealed that ligation of CD40 on EC promoted an increased association of Ras with its effector molecules Raf, Rho, and phosphatidylinositol 3-kinase (PI3K). But, cotransfection of effector-loop mutants of Ras determined that only PI3K was functional for Ras-induced VEGF transcription. Also, wortmanin and a dominant-inhibitory mutant of PI3K inhibited CD40-induced overexpression of VEGF. Together these findings demonstrate that both Ras and PI3K are intermediaries in CD40-induced regulation of VEGF in EC. We believe our findings are of importance in several chronic inflammatory diseases, including atherosclerosis and allograft rejection associated with both CD40-CD40 ligand signaling as well as VEGF expression and function.

CD40-induced transcriptional activation of vascular endothelial growth factor involves a 68-bp region of the promoter containing a CpG island.

Vascular endothelial growth factor (VEGF) is produced by several cell types in the kidney, and its expression is tightly regulated for the maintenance of normal renal physiology. Increases or decreases in its expression are associated with proteinuria and renal disease. Recently, we found that the expression of VEGF is markedly induced following interactions between CD40 ligand (CD40L) and CD40. Here, endothelial cells (EC) or Jurkat T cell lines were transiently transfected with luciferase reporter constructs under the control of the human VEGF promoter and were treated with human soluble CD40L (sCD40L). We identified a CD40-responsive 68-bp region (bp -50 to +18) of the promoter and 43 bp within this region (bp -25 to +18) that have 97% homology to a sequence of CpG dinucleotides. A computerized search revealed that the CpG region has putative binding domains for the transcriptional repressor protein methyl CpG binding protein-2 (MeCP2). In EMSA, we found that the 43-bp methylated sequence formed four complex(es) with nuclear extracts from untreated EC and reduced binding of at least one complex when nuclear lysates from sCD40L-activated EC (30 min) were used. Supershift analysis using anti-MeCP2 demonstrated that most of the complex(es) in both untreated and sCD40L-activated EC involved interactions between the 43-bp DNA and MeCP2. In addition, we found that other CpG binding proteins may also interact with this region of the promoter. Taken together, this is the first demonstration that CpG binding transcriptional repressor proteins including MeCP2 may be of importance in VEGF biology.

Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette transporter.

Cell fusion involving progenitor cells is a newly recognized phenomenon thought to contribute to tissue differentiation. The molecular mechanisms governing cell fusion are unknown. P-glycoprotein and related ATP-binding cassette transporters are expressed by progenitor cells, but their physiological role in these cell types has not been defined. Here, we have cloned ABCB5, a rhodamine efflux transporter and novel member of the human P-glycoprotein family, which marks CD133-expressing progenitor cells among human epidermal melanocytes and determines as a regulator of membrane potential the propensity of this subpopulation to undergo cell fusion. Our findings show that polyploid ABCB5+ cells are generated by cell fusion and that this process is specifically enhanced by ABCB5 P-glycoprotein blockade. Remarkably, multinucleated cell hybrids gave rise to mononucleated progeny, demonstrating that fusion contributes to culture growth and differentiation. Thus, our findings define a molecular mechanism for cell fusion involving progenitor cells and show that fusion and resultant growth and differentiation are not merely spontaneous events, but phenomena regulated by ABCB5 P-glycoprotein.