The distinct metabolic phenotype of lung squamous cell carcinoma defines selective vulnerability to glycolytic inhibition.

Adenocarcinoma (ADC) and squamous cell carcinoma (SqCC) are the two predominant subtypes of non-small cell lung cancer (NSCLC) and are distinct in their histological, molecular and clinical presentation. However, metabolic signatures specific to individual NSCLC subtypes remain unknown. Here, we perform an integrative analysis of human NSCLC tumour samples, patient-derived xenografts, murine model of NSCLC, NSCLC cell lines and The Cancer Genome Atlas (TCGA) and reveal a markedly elevated expression of the GLUT1 glucose transporter in lung SqCC, which augments glucose uptake and glycolytic flux. We show that a critical reliance on glycolysis renders lung SqCC vulnerable to glycolytic inhibition, while lung ADC exhibits significant glucose independence. Clinically, elevated GLUT1-mediated glycolysis in lung SqCC strongly correlates with high 18F-FDG uptake and poor prognosis. This previously undescribed metabolic heterogeneity of NSCLC subtypes implicates significant potential for the development of diagnostic, prognostic and targeted therapeutic strategies for lung SqCC, a cancer for which existing therapeutic options are clinically insufficient.

Proteomic analysis of human meibomian gland secretions.

BACKGROUND/AIM: Human tears contain hundreds of proteins that may exert a significant influence on tear film stability, ocular surface integrity, and visual function. The authors hypothesise that many of these proteins originate from the meibomian gland. This study's aim was to begin to develop the proteomic methodology to permit the testing of their hypothesis. METHODS: Meibomian gland secretions were collected from the lower eyelids of adult volunteers and placed in a chloroform-methanol mixture. Samples were partitioned in a biphasic system and non-lipid phase materials were reduced, alkylated, and trypsin digested to obtain peptides for protein identification. This peptide mixture was separated by micro-capillary reverse phase chromatography and the effluent examined by nano-electrospray MS and data dependent MS/MS. SEQUEST software was used to identify proteins from the MS/MS spectra. RESULTS: The methodological approach to date has permitted the identification of more than 90 proteins in human meibomian gland secretions. Proteins include the alpha2-macroglobulin receptor, IgA alpha chain, farnesoid X activated receptor, interferon regulatory factor 3, lacritin precursor, lactotransferrin, lipocalin 1, lysozyme C precursor, potential phospholipid transporting ATPase IK, seven transmembrane helix receptor (also termed somatostatin receptor type 4), testes development related NYD-SP21 (also termed high affinity IgE receptor beta subunit), and TrkC tyrosine kinase. CONCLUSIONS: These findings indicate that the meibomian gland secretes a number of proteins into the tear film. It is quite possible that these proteins contribute to the dynamics of the tear film in both health and disease.

HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity.

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

Effect of androgen deficiency on the human meibomian gland and ocular surface.

The purpose of this study was to determine whether the chronic use of antiandrogen medications leads to meibomian gland dysfunction, altered lipid profiles in meibomian gland secretions, decreased tear film stability, and evaporative dry eye. Subjects taking antiandrogen therapy for prostatic indications, as well as age-related controls, were asked to complete a questionnaire that assessed dry eye symptoms and then were given a complete anterior segment examination. Moreover, meibomian gland secretions were obtained from each eye and analyzed by high-performance liquid chromatography/mass spectrometry for the relative content of cholesterol, cholesterol esters, wax esters, diglycerides, triglycerides, and specific molecular species in the diglyceride fraction. Our results demonstrate that patients taking antiandrogen treatment, compared with age-related controls, had a: 1) significant increase in the frequency of appearance of tear film debris, an abnormal tear film meniscus, irregular posterior lid margins, conjunctival tarsal injection, and orifice metaplasia of the meibomian glands; 2) significant increase in the degree of ocular surface vital dye staining; 3) significant decrease in the tear film breakup time and quality of meibomian gland secretions; and 4) significant increase in the frequency of light sensitivity, painful eyes, and blurred vision. In addition, the use of antiandrogen pharmaceuticals was associated with significant changes in the relative amounts of lipids in meibomian gland secretions. Our findings indicate that chronic androgen deficiency is associated with meibomian gland dysfunction and dry eye.

Immune response to intragraft antigen in draining lymph nodes after corneal transplantation is mediated by interleukin-12.

To examine the molecular basis of immunity generated to intragraft antigens and determine whether it differs between acceptor and rejector hosts, we used a novel in vitro system to assay the T cell response to a specific antigen, ovalbumin (OVA), in the graft. OVA-containing corneas were orthotopically grafted into syngeneic or allogeneic hosts. Draining cervical lymph nodes (cLN) were assayed for OVA-specific T cell proliferation and cytokine production. In addition, cytokine production was assayed in cultures of antigen-presented cells (APC) isolated from cLN cultured with OVA-specific DO11.10 T cells and OVA. OVA-specific immunity was detected only in the draining cLN of mice following allogeneic, but not syngeneic, grafting, and this immunity was evident well before any demonstrable alloresponse in the graft. In addition, cLN cells from mice that accepted their corneal allografts produced significantly less interferon-gamma (IFN-gamma) when stimulated in culture than cells harvested from cLN of rejector hosts. Moreover, APC isolated from cLN of acceptor hosts produced significantly lower levels of IL-12. These data suggest that the induction of immunity to corneal antigens in the draining cLN occurs via an interleukin-12 (IL-12) and IFN-gamma-dependent mechanism. Targeting this process may serve as an effective immunomodulatory strategy in corneal transplantation.

Early expression of proinflammatory cytokines interleukin-1 and tumor necrosis factor-alpha after corneal transplantation.

This study's aim was to determine the early postoperative expression of proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) by corneal grafts. BALB/c (n = 90) and C57BL/6 (n = 90) murine recipients were grafted with donor corneas from either syngeneic or allogeneic mice. At 7 and 14 days after surgery, corneal grafts were excised and the recipient rims separated from the donor tissue. Corneal segments were cultured and assayed for cytokines by enzyme-linked immunosorbent assay (ELISA). There was profound upregulation in expression of both IL-1alpha and TNF-alpha after corneal transplantation. Among both low-rejecting BALB/c and high-rejecting C57BL/6 hosts, levels of IL-1alpha were significantly (p < 0.01) more marked in allogeneic as compared to syngeneic grafts. TNF-alpha overexpression was similarly more marked in allogeneic as compared to syngeneic grafts in both BALB/c and C57BL/6 hosts, although the difference was generally more marked among high-rejecting C57BL/6 recipients. In the case of both IL-1alpha and TNF-alpha, the principal source of cytokine expression in the transplanted tissue was the recipient rim. There is significant overexpression of both IL-1alpha and TNF-alpha during the first 2 weeks after transplantation in both syngeneic and allogeneic orthotopic corneal grafts. However, whereas in syngeneic grafts cytokine expression generally decreases after the first postoperative week, significantly elevated cytokine levels are sustained in allogeneic grafts, implicating IL-1 and TNF-alpha as mediators of the alloimmune response in corneal transplantation.

Molecular mechanisms of immunity in corneal allotransplantation and xenotransplantation.

Corneal allotransplantation is the most common and successful form of solid organ transplantation in humans. In uncomplicated cases, the two-year graft survival rate is over 90%. This extraordinary success can be attributed in part to various features of the normal cornea and anterior segment that together account for their 'immune-privileged' status. However, despite this success, a significant number of corneal grafts fail and immunological rejection remains by far the leading cause of graft failure. Studies on animal models of corneal transplantation have yielded a wealth of information on the molecular and cellular features of graft rejection, and have established that this process is mediated primarily by CD4+ T cells of the T helper 1 (Th1) phenotype. In addition, studies have elucidated that certain facets of allosensitisation differ between corneal and other solid organ transplants. On the basis of these findings, novel experimental strategies selectively targeting the afferent or efferent arms of corneal alloimmunity have provided promising results in preventing corneal allograft rejection in the laboratory. Finally, because of the global shortage of human donor corneas, there is currently renewed interest in the possibility of using corneas from other species for transplantation into human eyes (xenotransplantation). Preliminary studies on animal models of corneal xenotransplantation have documented both antibody-mediated and cell-mediated responses that might play important roles in the accelerated rejection observed in corneal xenotransplants. This review synthesises the principal concepts emerging from studies of the molecular mechanisms in corneal transplant immunology.

Topical interleukin 1 receptor antagonist promotes corneal transplant survival.

BACKGROUND: Interleukin (IL)-1 is a potent proinflammatory cytokine that plays a critical role in initiating and maintaining immunogenic inflammation. We performed a series of experiments to determine whether the topical application of IL-1 receptor antagonist (IL-1ra) can prolong corneal transplant survival in the murine model of orthotopic allotransplantation. METHODS: For all experiments, C57BL/6 corneas were transplanted into BALB/c (major histocompatibility and minor histocompatibility-disparate) eyes. "High-risk" transplants were transplants that had been sutured into BALB/c recipient beds with corneal neovascularization induced by placement of three interrupted sutures in the host cornea 2 weeks earlier. Both risk groups were divided in a masked fashion into treatment subgroups that received either 20 mg/ml of IL-1ra mixed in 0.2% sodium hyaluronate vehicle (n=28) or placebo alone (n=25). All transplants were evaluated for 8 weeks after surgery for signs of rejection. At the end of follow-up, corneal specimens were processed for enumeration of Langerhans cells and histopathological evaluation. RESULTS: Survival rates of both normal-risk and high-risk transplants increased significantly among the IL-1ra-treated animals compared with untreated controls by both stratified Mantel-Haenszel (P=0.02) and Kaplan-Meier survival (P=0.03) analyses. Furthermore, both normal- and high-risk IL-1ra-treated grafts had significantly less inflammation and Langerhans cells infiltration compared with untreated controls. CONCLUSIONS: Topical treatment with IL-1ra has a significantly positive effect in promoting corneal allograft survival.

Role of tumor necrosis factor receptors TNFR-I (P55) and TNFR-II (P75) in corneal transplantation.

BACKGROUND: To determine the role of tumor necrosis factor-alpha (TNF-alpha) receptor (TNFR) function in corneal allograft immunology. METHODS: Animals with gene-targeted deficiency in TNFR-I (p55-/-), TNFR-II (p75-/-), or combined TNFR-I/TNFR-II deficiency (p55-/-p75-/-) and their wild-type controls were used as recipients of fully-mismatched (BALB/c; n=88) or multiple minor alloantigen-mismatched (BALB.b; n=62) orthotopic corneal transplants to determine the effect of selective deficiency in one or both TNF-alpha receptors on corneal allograft survival. Grafted recipients were followed biomicroscopically for signs of rejection, and survival data were analyzed by the Kaplan-Meier method. RESULTS: There was no discernible difference in survival of fully-mismatched BALB/c corneal grafts in p55-/- (n=12; P=0.76) or in double-knockout p55-/-p75-/- (n=13; P=0.41) as compared with wild-type C57BL/6.129 hosts. However, the survival of BALB/c allografts was lower in p75-/- (n=10; median survival 20 days) as compared with control C57BL/6 (n=30; median survival 30 days) hosts (P=0.02). In contrast, there was no discernible effect in survival of minor alloantigen-disparate BALB.b corneal grafts in p75-/- (n=13; P=0.95) or in combined p55-/-p75-/-(n=10; P=0.17) hosts as compared with C57BL/6 (n=9) and C57BL/6.129 (n=10) wild-type controls, respectively. However, there was a profound enhancement in the survival of BALB.b allografts in p55-/- recipients (n= 10; median survival 35 days) as compared to wild-type C57BL/6.129 (n=10; median survival 25 days) controls (P<0.01). CONCLUSIONS: Our data suggest that the two TNF-alpha receptors largely play discrete roles in mediating rejection of murine corneal allografts. TNFR-I (p55) function seems to be integral to the rejection of minor-disparate grafts, and its selective suppression leads to enhancement of allograft survival. In contrast, TNFR-II (p75) function appears to be associated with enhanced survival of major histocompatibility complex-disparate allografts. The combined deletion of TNFR functionality in p55-/-p75-/- confers no net advantage or disadvantage to major histocompatibility complex or minor alloantigen-disparate grafts.

ICAM-1 deficiency suppresses host allosensitization and rejection of MHC-disparate corneal transplants.

BACKGROUND: We used a murine model of orthotopic corneal transplantation to determine whether host deficiency in ICAM-1 promotes survival of corneal grafts with different degrees of allodisparity. METHODS: ICAM-1-/- and wild-type C57BL/6 (ICAM-1+/+) received corneal grafts from the following strains of mice: BALB/c (fully mismatched), BALB.b (mismatched at multiple minor H only), or B10.D2 [including major histocompatibility complex (MHC) mismatch]. Graft rejection, induction of allospecific delayed-type hypersensitivity (DTH) responses, and leukocytic infiltration of grafts were measured. RESULTS: There were no differences in long-term survival of allografts that were either fully mismatched or had only minor H disparity in ICAM-1+/+ vs. ICAM-1-/-hosts. However, whereas B10.D2 grafts were accepted in only 58% of the ICAM-1+/+ hosts, graft survival in ICAM-1-/- recipients was 100% (P=0.006). Moreover, none of the ICAM-1-/- mice receiving B10.D2 grafts developed allospecific DTH. CONCLUSIONS: Prolonged survival seen in MHC-mismatched grafts in ICAM-1-/- mice, along with a suppressed DTH response to donor alloantigens after transplantation, suggest that ICAM-1 is associated with recipient sensitization to MHC alloantigens.

Loss and restoration of immune privilege in eyes with corneal neovascularization.

PURPOSE: To delineate the time course for loss of immune privilege after induction of corneal neovascularization (NV), and to test whether treatment with angiostatic agents can restore the eye's capacity to induce anterior chamber-associated immune deviation (ACAID). METHODS: Corneal NV in murine eyes was induced by placement of intrastromal sutures. At different time points after NV induction, study eyes were initiated on a 10-day regimen of one of a variety of anti-inflammatory or angiostatic agents. After the completion of their treatment regimen, eyes were tested as to whether they could support ACAID. To test whether any observed effect on the delayed-type hypersensitivity response was because of a systemic absorption of the topically applied medication, certain animals had only their fellow eyes treated. RESULTS: Inflammatory corneal NV leads to the loss of immune privilege during the first week of the NV induction. Left untreated, these eyes remain incapable of supporting ACAID, even weeks after the initial corneal insult. However, when treatment with an anti-inflammatory agent is initiated during the first 2 weeks after the NV induction, these eyes show a restored capacity for ACAID induction, and this appears to be unrelated to any systemic effect of the treatment regimen. Treatment started at later time points is not capable of restoring the eye's normal capacity for inducing deviant immunity. CONCLUSIONS: Corneal neovascularization leads to loss of immune privilege in the anterior segment manifested as the inability to sustain ACAID. Moreover, topical angiostatic strategies can lead to restoration of immune privilege when instituted sufficiently early in the course of the neovascular response.

The critical role of lymph nodes in corneal alloimmunization and graft rejection.

PURPOSE: To elucidate the role of draining cervical lymph nodes (CLNs) in corneal alloimmunity. METHODS: Fully mismatched orthotopic corneal transplantation was performed in BALB/c hosts that had their CLNs excised before transplantation (CLN(-)). Normal hosts (CLN(+)), splenectomized mice (Sp(-)), and those without either CLNs or spleen (CLN(-)/Sp(-)) served as comparison groups. To determine the contribution of CLNs to alloimmunity more directly, CLN(-) mice were reconstituted by grafting LNs from other BALB/c mice to their cervical lymphatic chains, thus deriving CLN(-/+) mice. Tetramethyl rhodamine isothiocyanate's (TRITC) flow to draining CLNs was used as a measure of afferent lymph flow. Graft survival and allospecific delayed-type hypersensitivity (DTH) were used as measures of alloreactivity. RESULTS: Fifty percent of normal control and 12% of Sp(-) hosts accepted the allografts. In contrast, 100% of CLN(-) and 88% of CLN(-)/Sp(-) hosts accepted allografts indefinitely (P < 0.01). Additionally, all CLN(-) hosts failed to demonstrate allospecific DTH (P < 0.001). CLN(-/+) mice reconstituted with LN from naïve animals showed graft survival rates and DTH responses that were indistinguishable from those of naïve CLN(+) mice. Of particular interest, however, is that mice reconstituted with CLNs from hosts with rejected corneal grafts had swift rejection of subsequent corneal grafts and exhibited strong donor-specific DTH. In contrast, mice reconstituted with CLNs from hosts with accepted corneal grafts showed rejection of subsequent corneal grafts in a manner that was indistinguishable from rejection in naïve CLN(+) hosts. CONCLUSIONS: Draining CLNs play a critical role in allosensitization and rejection. In contrast to the spleen, draining CLNs do not appear to play a critical role in tolerance induction in corneal transplantation.

Expression of cell adhesion molecules on limbal and neovascular endothelium in corneal inflammatory neovascularization.

PURPOSE: To investigate the expression of cell-adhesion molecules on corneolimbal and neovascular endothelium and the associated leukocyte infiltration in an experimental model of inflammatory corneal neovascularization (NV). METHODS: Corneal NV was induced in BALB/c mice by placement of nylon sutures. Interleukin-1 receptor antagonist (IL-1ra) was used topically to determine whether suppression of IL-1 could affect adhesion molecule expression and leukocytic infiltration. At set time points, corneal samples were analyzed immunohistochemically for expression of P-selectin, E-selectin, intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, and platelet- endothelial adhesion molecule (PECAM)-1. Leukocytic infiltration at different time points was quantified histologically. In companion experiments mice deficient in ICAM-1 were investigated to determine the functional relevance of this molecule in corneal leukocyte infiltration. RESULTS: Significant enhanced expression of ICAM-1 was detected on the corneolimbal vascular endothelium as early as 8 hours and on the newly formed corneal NV by day 3, and treatment with IL-1ra led to significant suppression of this expression. IL-1ra-induced suppression of ICAM-1 expression was accompanied by a profound decrease in corneal leukocytic infiltration by 44.6% at day 1 (P < 0.003), 71.8% at day 3 (P < 0.001), 60.1% at day 7 (P < 0.001), and 63.8% at day 14 (P < 0.001), compared with control corneas. Similarly, in ICAM-1 knockout mice, the corneal leukocytic infiltration was 50.3%, 52.9%, and 36.4%, compared with wild-type control animals on day 1 (P < 0.001), day 7 (P < 0.005), and day 14 (P < 0.001), respectively. Expression of PECAM-1 was constitutively present on perilimbal vascular endothelium and had no response to IL-1ra treatment. No significant expression of P-selectin, E-selectin, or VCAM-1 was detected in this experimental model. CONCLUSIONS: These results suggest that leukocytic infiltration in this model of inflammatory corneal NV is closely associated with ICAM-1 expression, and that topical IL-1ra displays corneal anti-inflammatory effects, largely by suppressing ICAM-1 expression on vascular endothelial cells.

Blockade of CD40-CD154 costimulatory pathway promotes survival of allogeneic corneal transplants.

PURPOSE: To determine the effect of systemic anti-CD154 monoclonal antibody on the survival of orthotopic murine corneal transplants. METHODS: BALB/c mice were used as recipients of syngeneic, multiple minor histocompatability (H)-disparate, or major histocompatibility complex MHC-mismatched corneal transplants. Recipient beds were either avascular (normal risk) or neovascularized (high risk). Mice were randomized to receive either anti-CD154 antibody or control immunoglobulin by intraperitoneal injection at surgery and once weekly after surgery. After orthotopic corneal transplantation, all grafts were evaluated for signs of rejection by slit lamp biomicroscopy over 8 weeks. The high-risk transplants were continuously observed until week 18 after the therapy was discontinued at week 8. Allospecific delayed-type hypersensitivity (DTH) was evaluated after transplantation in high-risk graft recipients. Frequency of interferon (IFN)-gamma-secreting T cells in the hosts was measured by enzyme-linked immunospot (ELISPOT) assay. RESULTS: In normal-risk transplantation, the 8-week survival rate improved from 25% in control mice to 88% in anti-CD154-treated hosts of minor H-disparate grafts (P = 0.0087) and from 78% in control mice to 100% in anti-CD154-treated recipients of MHC-mismatched transplants (P = 0.177). Of particular significance, in high-risk transplantation, anti-CD154 therapy dramatically enhanced the survival of both minor H- and MHC-disparate corneal transplants to 100% (P = 0.0001) and 92% (P = 0.0002), respectively. In addition, the anti-CD154-treated mice did not exhibit allospecific immunity. However, termination of anti-CD154 led to some loss in graft survival, especially among high-risk minor H-disparate grafts. The frequency of IFN-gamma-producing T cells was significantly reduced in anti-CD154-treated hosts. CONCLUSIONS: Continuous suppression of the CD40-CD154 costimulatory pathway promotes the acceptance of corneal transplants, regardless of the degree of allodisparity or preoperative risk. The beneficial effect of anti-CD154 treatment may be due in part to inhibition of Th1-mediated responses.

Interleukin-1 receptor antagonist therapy and induction of anterior chamber-associated immune deviation-type tolerance after corneal transplantation.

PURPOSE: Topical treatment with interleukin 1 receptor antagonist (IL-1ra) can promote corneal allograft survival by suppressing induction of allodestructive immunity. The purpose of these experiments was to determine whether IL-1ra could also promote induction of allo-protective tolerogenic pathways, including anterior chamber-associated immune deviation (ACAID), which has been shown to participate in long-term survival of corneal transplants. METHODS: Corneal buttons from BALB/c (syngeneic) or C57BL/6 (fully mismatched allogeneic) mice were orthotopically grafted onto BALB/c recipients. Topical IL-1ra or vehicle alone was applied to grafts three times daily. Donor-specific ACAID was measured in allogeneic grafted mice at 4 and 8 weeks after transplantation by ear-challenging grafted hosts with donor-derived splenocytes 1 week after SC immunization. In separate experiments, grafted mice were treated for 4 weeks before injecting ovalbumin (OVA) into their anterior chambers to determine their capacity to induce antigen-specific ACAID. RESULTS: Treatment with IL-1ra did not promote, or inhibit, induction of donor-specific ACAID compared with vehicle-treated controls at either the early or late time points studied. However, IL-1ra treatment after transplantation led to significantly earlier restoration of the grafted eyes' capacity for inducing ACAID to soluble antigen (OVA). CONCLUSIONS: Promotion of OVA-specific ACAID by IL-1ra suggests that suppression of IL-1-mediated mechanisms contributes to recovery of the anterior segment's immunosuppressive microenvironment at least 1 month earlier than would otherwise be seen after corneal transplantation. However, IL-1ra treatment does not alter induction of donor-specific ACAID after transplantation, suggesting that its anti-inflammatory activities do not lead to an ACAID-inducing signal per se. This suggests that IL-1ra promotes graft survival almost exclusively by virtue of suppressing inflammation and not by directly promoting tolerance or antigen-specific regulatory pathways.

Differential chemokine gene expression in corneal transplant rejection.

PURPOSE: To evaluate the differential gene expression of chemokines after corneal transplantation and to determine the chemokines associated with allograft rejection. METHODS: Orthotopic mouse corneal transplantation was performed in two fully mismatched-strain combinations using C57BL/6 (H-2b) and BALB/c (H-2d) mice as recipients and BALB/c and C57BL/6 mice as donors. Normal nonsurgical eyes served as negative control specimens and syngeneic transplants (isografts) as control specimens for the alloimmune response. Chemokine gene expression in accepted and rejected allografts and appropriate control specimens was determined by a multiprobe RNase protection assay system. RESULTS: In eyes with rejected allografts, there was overexpression of regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, and monocyte chemotactic protein (MCP)-1 in both C57BL/6 and BALB/c recipients. In addition, C57BL/6 eyes with rejected allografts expressed very high levels of interferon-gamma-inducible protein of 10 kDa (IP-10) mRNA, in contrast to BALB/c eyes with rejected allografts, in which IP-10 expression remained very low. In contrast, lymphotactin gene expression increased only slightly in rejected allografts, and eotaxin mRNA, which was also detected in normal eyes, remained unchanged among isograft and allograft groups. T-cell activation gene (TCA)-3 mRNA was not detected in any of the assayed eyes. CONCLUSIONS: Increased expression of mRNA for select chemokines of the CXC (alpha) and CC (beta) families is associated with corneal allograft rejection. Significantly elevated IP-10 gene expression in high-rejector C57BL/6, but not in low-rejector BALB/c, hosts suggests that differential activation of chemokines may be related to differences in alloimmune reactivity observed among different murine strains.

Low-dose ultraviolet-B irradiation of donor corneal endothelium and graft survival.

Donor rabbit corneal endothelium was pretreated with different doses of ultraviolet (UV-B) irradiation (302 nm) before grafting to test whether allograft survival could be favorably affected in comparison with untreated corneas grafted into the same recipients. Endothelial rejection was observed in 19 of 32 (59%) eyes that received no treatment compared with five of 32 (16%) eyes that received UV-B (P less than 0.001), and increasing doses of UV-B were associated with lower rejection rates (P less than 0.05). Although exposure of donor endothelium significantly reduced endothelial rejection at all doses tested, it resulted in primary graft failure in a substantial proportion of corneas treated at high doses. Class II (Ia) antigen staining of corneal tissue was present in conjunction with clinical evidence of rejection, and the magnitude of staining correlated with the histologic extent of inflammation. Scanning electron microscopy revealed various endothelial cell surface irregularities and membrane defects in high-dose UV-treated corneas. Endothelial cell cultures exposed in vitro to UV-B light showed a dose-dependent loss in cell viability. These data suggest that UV-B pretreatment of donor corneal endothelium prolongs graft survival but that toxic side effects must be carefully controlled.

Interleukin-1 receptor antagonist suppresses Langerhans cell activity and promotes ocular immune privilege.

PURPOSE: To determine whether the capacity of Langerhans cells (LCs) to abrogate ocular immune privilege can be suppressed by the topical application of interleukin-1 receptor antagonist (IL-1ra). METHODS: Cautery was applied to corneas of BALB/c mice on day 0 to induce centripetal migration of LCs. Immune privilege was tested by the ability to induce anterior chamber-associated immune deviation (ACAID) to intracamerally injected soluble antigen 1 to 2 weeks after cautery application. The number of LCs was enumerated by immunofluorescent staining. In other experiments, freshly procured Thy-1-depleted epidermal cells, with or without LC depletion, were injected directly into virgin murine corneas before testing for ACAID. All test animals were randomized for treatment with either topical IL-1ra or placebo in a masked fashion for 1 to 2 weeks after induction of LC migration and before intracameral injection of antigen. RESULTS: Intracorneal injection of freshly procured LC-depleted epidermal cells into normal eyes failed to abrogate ACAID, whereas LC-containing cell populations uniformly led to loss of immune privilege (P < 0.01). Topical treatment with IL-1ra led to retention of the cauterized eyes' capacity for ACAID induction (P < 0.01) and to a profound (>80%) suppression of LC migration compared with untreated controls (P < 0.01). Additionally, topical IL-1ra treatment of eyes with intracorneally injected LCs preserved immune privilege and ACAID induction (P < 0.001). CONCLUSIONS: IL-1 mediates mechanisms of immunity in corneal inflammation that subvert the normal eye's immune privileged state. However, its antagonism with topical administration of IL-1ra preserves ocular immune privilege and ACAID through suppression of LC function.

Prevention of allergic eye disease by treatment with IL-1 receptor antagonist.

PURPOSE: To determine the impact of interleukin-1 (IL-1) inhibition using IL-1 receptor antagonist (IL-1Ra) in a mouse model of allergic eye disease. METHODS: A/J mice sensitized and challenged with cat dander in the eye were treated with topical IL-1Ra or vehicle alone. Control mice were treated with IL-1Ra or vehicle but sensitized and challenged with phosphate-buffered saline alone. Immediately after the final allergen challenge, the mice were observed for behavioral changes and assessed for lid injection and chemosis. The animals were then killed, eyes and attached lids were removed for either RNA extraction or histology, and draining lymph nodes were removed for either RNA extraction or in vitro stimulation assays. Differences in chemokine message between experimental and control groups-were determined by RNase protection assays. RESULTS: Treatment with IL-1Ra in allergen-challenged animals significantly reduced allergen-induced changes in photosensitivity (60%, P = 0.0002), chemosis (50%, P = 0.0151), and injection (86.7%, P = 0.0068) compared with vehicle-treated controls. Interleukin-1Ra reduced the number of degranulated mast cells and caused a significant reduction in the number of eosinophils infiltrating the conjunctival matrix (P<0.001) after allergen challenge. Examination of chemokine mRNA taken from the conjunctiva and draining lymph nodes by RNase protection assay showed a profound decrease in the production of a number of C-C chemokines. CONCLUSIONS: These findings suggest that IL-1Ra is suppressing allergic eye disease by a down-modulation of the recruitment of eosinophils and other inflammatory cells essential for the immunopathogenesis of ocular atopy.

Corneal transplantation in antibody-deficient hosts.

PURPOSE: To examine the role of donor-specific antibodies, with or without complement, in rejection of orthotopic corneal transplants by using mice as recipients in which the genes for the heavy chain of immunoglobulin or the third complement component have been eliminated by homologous recombination. METHODS: BALB/c corneas were transplanted into eyes of B-cell-deficient (n=17) or wild-type control C57BL/6 (n=30) mice and into eyes of complement (C3)-deficient (n=15) or wild-type control 129-C57BL/6 (n=13) mice. After surgery all grafts were evaluated over 8 weeks in a masked manner by biomicroscopy for signs of rejection. RESULTS: The rates of corneal transplant rejection were similar among B-cell-deficient and C3-deficient mice compared with rejection rates in their respective wild-type control subjects. This similarity applied to the time course of rejection and to cumulative survival rates. CONCLUSIONS: Neither donor-specific antibody nor the third component of complement play essential roles in acute rejection of orthotopic corneal allografts in mice.