Meredith S. Gregory

Fas Ligand Engagement of Resident Peritoneal Macrophages In Vivo Induces Apoptosis and the Production of Neutrophil Chemotactic Factors

Fas ligand (FasL) is a potent proapoptotic type-II transmembrane protein that can cause cell death in Fas+ target populations. Despite the presumed “silent” nature of apoptotic cell death, forced expression of FasL can induce a dramatic inflammatory response. To elucidate the in vivo mechanism(s) linking FasL and inflammation, we used a membrane-bound cell-free form of FasL (mFasL-vesicle preparation (VP)). We found that i.p. injection of FasL-microvesicles led to the rapid activation and subsequent demise of Mac1high resident peritoneal macrophages. Apoptosis of Mac1high peritoneal macrophages was observed within 0.5 h of mFasL-VP injection and correlated with the detection of increased macrophage inflammatory protein (MIP)-2 levels in peritoneal lavage fluid as well as induced RNA expression of IL-1β, MIP-2, MIP-1α, and MIP-1β. In vitro culture of purified peritoneal populations identified Mac1high cells as the major cytokine/chemokine producers in response to mFasL-VP. Purified Mac1high cells exposed to FasL could restore the ability of Fas-deficient mice to mount an inflammatory response. Our data demonstrate that the FasL-mediated inflammatory response starts with the production of proinflammatory mediators by preapoptotic resident tissue macrophages and suggest a general mechanism responsible for neutrophil inflammation seen in cases of FasL-expressing allografts.

Effects of acute ethanol exposure on cellular immune responses in a murine model of thermal injury.

To test the effects of acute ethanol exposure on immune function after thermal injury, mice with blood alcohol levels of 100 mg/dL were given a 15% total body surface area dorsal scald or sham injury. Bacterial challenge resulted in 100 and 40% mortality in burn + ethanol‐ and burn + vehicle‐treated mice, respectively. Delayed‐type hypersensitivity responses were also significantly suppressed in burn + ethanol‐treated mice. At 1 and 4 days post‐burn, concanavalin A (ConA) ‐induced total splenocyte proliferation in burn + ethanol‐treated groups was significantly decreased (P < 0.01) compared with burn + vehicle‐ or sham‐treated animals. This decrease was not observed in total splenocytes cultured with anti‐CD3ε or among adherence‐depleted splenocytes given ConA or anti‐CD3ε. FACS analyses revealed no changes in splenocyte sub‐type ratios in burn + ethanol mice. The data herein demonstrate that acute ethanol exposure before thermal injury results in enhanced susceptibility to bacterial infection and markedly suppressed cellular immunity, which appears to be macrophage dependent. J. Leukoc. Biol. 62: 733–740; 1997.

ABCB5 is a limbal stem cell gene required for corneal development and repair

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

By Altering Ocular Immune Privilege, Bone Marrow-derived Cells Pathogenically Contribute to DBA/2J Pigmentary Glaucoma.

Pigment dispersion syndrome causes iris pigment release and often progresses to elevated intraocular pressure and pigmentary glaucoma (PG). Because melanin pigment can have adjuvant like properties and because the Gpnmb gene, which contributes to pigment dispersion in DBA/2J (D2) mice, is expressed in dendritic cells, we tested the hypothesis that ocular immune abnormalities participate in PG phenotypes. Strikingly, we show that D2 eyes exhibit defects of the normally immunosuppressive ocular microenvironment including inability of aqueous humor to inhibit T cell activation, failure to support anterior chamber (AC)-associated immune deviation, and loss of ocular immune privilege. Histologic analysis demonstrates infiltration of inflammatory leukocytes into the AC and their accumulation within the iris, whereas clinical indications of inflammation are typically very mild to undetectable. Importantly, some of these abnormalities precede clinical indications of pigment dispersal, suggesting an early role in disease etiology. Using bone marrow chimeras, we show that lymphohematopoietic cell lineages largely dictate the progression of pigment dispersion, the ability of the eye to support induction of AC-associated immune deviation, and the integrity of the blood/ocular barrier. These results suggest previously unsuspected roles for bone marrow–derived cells and ocular immune privilege in the pathogenesis of PG.

Bacterial endophthalmitis: Therapeutic challenges and host–pathogen interactions

Endophthalmitis is an infection of the posterior segment of the eye that frequently results in loss of vision. This devastating result occurs despite prompt and often aggressive therapeutic and surgical intervention. Over the past decade, research has centered on determining the bacterial and host factors involved in this potentially blinding disease. The initial focus on the bacterial factors responsible for intraocular virulence has recently expanded into analysis the inflammatory response to infection, including the molecular and cellular interactions between the pathogen and host. This review discusses the epidemiology and therapeutic challenges posed by endophthalmitis, as well as recent findings from the analysis of interactions between the host and pathogen. Based on these findings, a model for the pathogenesis of endophthalmitis is presented. A more comprehensive understanding of the molecular and cellular interactions taking place between pathogen and host during endophthalmitis will expose possible therapeutic targets designed to arrest the infection and prevent vision loss.

Estrogen regulation of immune responses after injury

There is a naturally occurring gender difference in immune responses which persists after traumatic injury. Physiological levels of 17beta-estradiol (E(2)) are immunostimulatory, whereas high pregnancy and superphysiological levels are immunosuppressive. In contrast, at all concentrations, testosterone suppresses immune responses. Evidence from this laboratory and others suggest that the gender difference in immune responses after injury is mediated in part by alterations in the circulating levels of gonadal steroid hormones through modulation of production of inflammatory and immunoregulatory cytokines, including interleukin-6 (IL-6). Aberrant production of IL-6 is known to be an important mediator of immunity after injury. Since E(2) is a critical regulator of IL-6 production and overall immune function, it suggests gender specific therapies should be considered for the treatment of patients.

Acute Ethanol Exposure Prior To Thermal Injury Results In Decreased T-cell Responses Mediated In Part By Increased Production Of Il-6

Previous studies by our laboratory have demonstrated that acute ethanol exposure prior to thermal injury results in suppression of cellular immune responses when compared with thermal injury alone. Ethanol exposure and burn injury are independently known to result in elevated IL-6, a cytokine with potent immunosuppressive properties. Therefore, we examined the role of IL-6 in the immune dysfunction in mice following a 15% body surface area scald (or sham) injury combined with acute ethanol (or vehicle) treatment. At 24 h post-injury, we observed slightly suppressed splenocyte proliferative responses and elevated circulating IL-6 (149 ± 15 pg/mL) in mice receiving burn alone compared with those receiving sham injury (31 ± 7 pg/mL). In contrast, burn + ethanol treated mice showed a profound suppression of splenocyte proliferation (20% of control) and significantly elevated circulating IL-6 levels (738 ± 218 pg/mL). The suppressed splenocyte proliferative response was found to be macrophage dependent. Furthermore, IL-6 production was significantly elevated (p < .05) in splenic macrophage cultures from burn + ethanol mice (159 ± 6 pg/mL) when compared with burn alone (109 ± 10 pg/mL). Treatment of the splenocyte cultures from burn + ethanol mice with an anti-IL6 monoclonal antibody resulted in partial restoration of splenocyte proliferation. Taken together, these data strongly suggest that the immune dysfunction observed in ethanol-exposed, thermally injured mice is mediated in part by elevated levels of IL-6.

Neutrophil chemokine production in the skin following scald injury

The present study was conducted to determine whether local production of neutrophil chemoattractant cytokines preceded the influx of neutrophils following dermal scald injury. To accomplish this, dermal tissue was examined for inflammatory infiltrate and the level of KC, a murine homolog of human interleukin-8, at various time points after scald injury. The studies reveal that there was a largely neutrophilic infiltrate at 1 day post-injury which persisted for 4 days. Dermal KC levels increased significantly at 4 h, returned to baseline at 8 h and were elevated again from 1 to 3 days post-burn (P < 0.01). At 3 days post-burn, KC was elevated 15-fold above the level in sham treated mice (P < 0.01). These observations demonstrate that the influx of neutrophils into the skin follows the expression of KC in the skin. This suggests that it should be possible to alter neutrophil accumulation at the wound site by manipulating the local chemokine signal.

Membrane Fas Ligand Activates Innate Immunity and Terminates Ocular Immune Privilege

It has been proposed that the constitutive expression of Fas ligand (FasL) in the eye maintains immune privilege, in part through inducing apoptosis of infiltrating Fas+ T cells. However, the role of FasL in immune privilege remains controversial due to studies that indicate FasL is both pro- and anti-inflammatory. To elucidate the mechanism(s) by which FasL regulates immune privilege, we used an ocular tumor model and examined the individual roles of the membrane-bound and soluble form of FasL in regulating ocular inflammation. Following injection into the privileged eye, tumors expressing only soluble FasL failed to trigger inflammation and grew progressively. By contrast, tumors expressing only membrane FasL 1) initiated vigorous neutrophil-mediated inflammation, 2) terminated immune privilege, and 3) were completely rejected. Moreover, the rejection coincided with activation of both innate and adaptive immunity. Interestingly, a higher threshold level of membrane FasL on tumors is required to initiate inflammation within the immune privileged eye, as compared with nonprivileged sites. The higher threshold is due to the suppressive microenvironment found within aqueous humor that blocks membrane FasL activation of neutrophils. However, aqueous humor is unable to completely block the proinflammatory effects of tumor cells that express high levels of membrane FasL. In conclusion, our data indicate that the function of FasL on intraocular tumors is determined by the microenvironment in conjunction with the form and level of FasL expressed.