Ascorbate in aqueous humor augments nitric oxide production by macrophages.

Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting proinflammatory NO production by macrophages (MΦs). Consistent with previous observations, we observed that although MΦs stimulated in the presence of AqH expressed NO synthase 2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure of NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of L-arginine (L-Arg) into L-citrulline, was augmented in lysates of MΦs stimulated in the presence of AqH. These data suggested that intracellular L-Arg may have been limited by AqH. However, we observed increased mRNA expression of the L-Arg transporter, cationic amino acid transporter 2B, and increased L-Arg uptake in MΦs stimulated in the presence of AqH. Arginases were expressed by stimulated Ms, but competition for L-Arg with NOS2 was excluded. Expression of GTP cyclohydrolase, which produces tetrahydrobiopterin (H(4)B), an essential cofactor for NOS2 homodimerization, increased after M stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together, these data provided no evidence for inhibited NOS2 enzymatic activity by AqH, suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH, and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in MΦs via ascorbate, which limited degradation of H(4)B. Therefore, AqH may augment NO production in macrophages by stabilizing H(4)B and increasing intracellular L-Arg.

S100A8/A9 proteins mediate neutrophilic inflammation and lung pathology during tuberculosis.

RATIONALE: A hallmark of pulmonary tuberculosis (TB) is the formation of granulomas. However, the immune factors that drive the formation of a protective granuloma during latent TB, and the factors that drive the formation of inflammatory granulomas during active TB, are not well defined. OBJECTIVES: The objective of this study was to identify the underlying immune mechanisms involved in formation of inflammatory granulomas seen during active TB. METHODS: The immune mediators involved in inflammatory granuloma formation during TB were assessed using human samples and experimental models of Mycobacterium tuberculosis infection, using molecular and immunologic techniques. MEASUREMENTS AND MAIN RESULTS: We demonstrate that in human patients with active TB and in nonhuman primate models of M. tuberculosis infection, neutrophils producing S100 proteins are dominant within the inflammatory lung granulomas seen during active TB. Using the mouse model of TB, we demonstrate that the exacerbated lung inflammation seen as a result of neutrophilic accumulation is dependent on S100A8/A9 proteins. S100A8/A9 proteins promote neutrophil accumulation by inducing production of proinflammatory chemokines and cytokines, and influencing leukocyte trafficking. Importantly, serum levels of S100A8/A9 proteins along with neutrophil-associated chemokines, such as keratinocyte chemoattractant, can be used as potential surrogate biomarkers to assess lung inflammation and disease severity in human TB. CONCLUSIONS: Our results thus show a major pathologic role for S100A8/A9 proteins in mediating neutrophil accumulation and inflammation associated with TB. Thus, targeting specific molecules, such as S100A8/A9 proteins, has the potential to decrease lung tissue damage without impacting protective immunity against TB.

CTL induction of tumoricidal nitric oxide production by intratumoral macrophages is critical for tumor elimination.

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment, tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors, whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms), suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However, CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically, the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer, tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly, in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion, the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress.

Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function.

We tested whether granulocytes, which contaminate PBMC isolates after prolonged blood storage at room temperature, are responsible for inhibited T cell function in aged blood. We extend previous observations by characterizing these contaminating granulocytes as CD11b+ CD15+ cells comparable to activated CD11b+ CD15+ granulocytes induced by incubation of blood with FMLP. Granulocyte contamination of PBMC was observed within 6-8 h after venipuncture and room temperature storage (2.3 fold increase), and increased 11.3-fold by 24-26 h in comparison to PBMC from fresh blood. Refrigerated 22-26 hour storage of blood exacerbated granulocyte contamination (84-fold increase). In contrast, granulocyte contamination was markedly reduced if blood was diluted in RPMI-1640 medium (3.9-fold increase) or PBS (1.8-fold increase) prior to 22-26 hour room temperature storage. Granulocyte contamination significantly correlated with reduced CD3zeta chain expression, a marker of T cell dysfunction. Correspondingly, T cell proliferation following PHA stimulation was significantly decreased in PBMC with contaminating granulocytes from aged blood (77% of control) or FMLP treated blood (44% of control). Minimizing granulocyte contamination in PBMC of aged blood by cell sorting, or by reducing granulocyte activation by diluting blood in PBS prior to storage, increased CD3zeta chain expression and increased T cell proliferation following stimulation. These data indicate that granulocytes inhibit T cell function in aged blood. Therefore, preventing granulocyte activation in blood specimens is critical to maintain optimal T cell function. This may be accomplished by limiting the time from venipuncture to PBMC isolation to <8 h and may be extended to 26 h by simply diluting blood in PBS prior to room temperature storage.

Use of superparamagnetic microbeads in tracking subretinal injections.

PURPOSE: The purpose of these studies was to develop a method to track intraocular injections. METHODS: Retinal pigment epithelial (RPE) cells, purified from adult mouse eyes, were incubated with superparamagnetic microbeads (Dynabeads, 4.5 microm) coated with bovine serum albumin to verify that they could phagocytose the microbeads. For in vivo tracking studies, mice were anesthetized and a small incision was made at the pars plana and 2 mul of microbeads (around 10(5) microbeads) or RPE that had taken up the microbeads were injected into the subretinal space (SRS). Mice were sacrificed at various times after injection. The eyes were enucleated, fixed in formalin, and embedded in paraffin. Sections were stained with H&E, visualized by light microscopy. Some eyes were digested with collagenase and inflammatory cells determined by flow cytometry. RESULTS: Cultured adult RPE phagocytosed the magnetic microbeads. One day after injection into the SRS, a retinal detachment was observed at the injection point and free microbeads were easily detected at this site. One week later, the host RPE cells had phagocytized the microbeads and the retina had reattached. No inflammatory response was detected in the eyes that were injected with microbeads in the SRS at any time examined. Histology showed normal morphology of all retinal layers around the injection site. The microbeads remained in situ throughout the study. CONCLUSIONS: Protein coated magnetic microbeads are non-inflammatory after injection into the SRS. Host RPE cells phagocytized the microbeads and the retina maintained a healthy morphology after reattachment. This technique proved not only to be a good training tool to determine the precise location of injection, but also provided a noninflammatory method for long term marking of delivery into the SRS. However, the microbeads can not be used as a tracer of injected RPE because the microbeads were readily transferred to the endogenous RPE.

CD8+ T-cell tolerance induced by delivery of antigen to the anterior chamber is not the result of de facto intravenous or mucosal administration of antigen.

PURPOSE: We tested whether antigen administration via the anterior chamber (a.c.) was equivalent to intravenous (i.v.) or mucosal administration antigen. METHODS: Ovalbumin (OVA)-specific CD8(+) T cells (OT-I) were enumerated in lymphoid tissues of C57Bl/6 (B6) mice via adoptive transfer after the same amount of antigen was administered via a.c., i.v., or mucosal routes. Lytic activity was measured in B6 and gammadeltaT cell-deficient B6 mice given OVA via a.c., i.v, or mucosal routes after injection with OVA in adjuvant. RESULTS: OVA a.c. induced a pattern of T-cell proliferation distinct from i.v. or mucosal administration. A.c. and i.v., but not mucosal, OVA induced cytolytic T lymphocyte (CTL) tolerance. The inhibition of CTL responses was significantly greater in mice given OVA a.c. rather than i.v. gammadeltaT cells contributed to a.c.-, but not i.v.-, induced CTL tolerance. CONCLUSIONS: A.c. administration of antigen not de-facto i.v. or mucosal administration of antigen.

Splenectomy restores tumoricidal activity to promote elimination of intraocular tumors.

We recently demonstrated that splenectomy restores an interaction between CD8+ T cells and macrophages necessary for intraocular tumor elimination. Taking into consideration other studies indicating that intraocular tumor growth does not induce tumor-specific CD8+ T-cell tolerance, our data suggest that splenectomy may influence the phenotype of tumor-associated macrophages.

Biology of advanced uveal melanoma and next steps for clinical therapeutics.

Uveal melanoma is the most common intraocular malignancy although it is a rare subset of all melanomas. Uveal melanoma has distinct biology relative to cutaneous melanoma, with widely divergent patient outcomes. Patients diagnosed with a primary uveal melanoma can be stratified for risk of metastasis by cytogenetics or gene expression profiling, with approximately half of patients developing metastatic disease, predominately hepatic in location, over a 15-yr period. Historically, no systemic therapy has been associated with a clear clinical benefit for patients with advanced disease, and median survival remains poor. Here, as a joint effort between the Melanoma Research Foundation's ocular melanoma initiative, CURE OM and the National Cancer Institute, the current understanding of the molecular and immunobiology of uveal melanoma is reviewed, and on-going laboratory research into the disease is highlighted. Finally, recent investigations relevant to clinical management via targeted and immunotherapies are reviewed, and next steps in the development of clinical therapeutics are discussed.

Ocular immune privilege and CTL tolerance.

The introduction of antigens into the anterior chamber (AC) of the eye, an immune-privileged site, induces immune responses that effectively eliminate ocular pathogens while minimizing tissue damage that can cause blindness. This specialized immune response, termed AC associated immune deviation (ACAID) is thought to be an evolutionary compromise to preserve the delicate microanatomy of the eye while maintaining ocular immune responses. The injection of soluble antigen in the AC of mice results in systemic tolerance characterized by reduced priming for antigen-specific delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses. Similarly, the injection of histo incompatible tumors into the AC of mice reduces priming for DTH responses specific to minor antigens. However, robust tumor-specific CTL responses are induced systemically following this treatment that are capable of eliminating a subsequent injection of the same tumors in the skin or the opposite eye. Interestingly, CTL responses induced by administration of tumors in the AC fail to eliminate the primary ocular tumor. In this review, we compare and contrast CTL responses generated by the injection of soluble or tumor-associated antigens in the AC and discuss mechanisms employed to induce ocular CTL tolerance.

Gammadelta T cells play an essential role in several forms of tolerance.

Gammadelta T cells were discovered in the mid-1980s, but the antigens they recognize and the biological functions they mediate are poorly understood. Although gammadelta T cells have the capacity to augment immunity to certain infections and kill certain tumor cells, they are generally not required for development of antibody responses, for graft rejection, or for development of autoimmune diseases. Nevertheless, gammadelta T cells accumulate at sites of inflammation induced by infection, tumor growth, and autoimmune lesions, where they have been shown to reduce the inflammatory reaction and tissue damage. In this review, we summarize the evidence that gammadelta T cells play an important role in the induction of various forms of tolerance.

Splenectomy promotes indirect elimination of intraocular tumors by CD8+ T cells that is associated with IFNγ- and Fas/FasL-dependent activation of intratumoral macrophages.

Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. CD8(+) T cells, IFNγ, and FasL, but not perforin, or TNFα were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNγ and FasL did not target tumor cells directly as the majority of SPLNX IFNγR1(-/-) mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNγR1. Bone marrow chimeras revealed that IFNγR1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mϕ) within intraocular tumors in an IFNγ- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNγ and Fas. As depletion of Mϕs limited CD8 T cell-mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNγ- and Fas/FasL-dependent activation of intratumoral Mϕs by CD8(+) T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis, and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interaction between CD8(+) T cells and Mϕs to limit the immunosurveillance of intraocular tumors.

Influence of CD8+ T regulatory cells on intraocular tumor development.

The interior of the eye, or uvea, is a site of immune privilege where certain immune responses are attenuated or completely excluded to protect non-regenerating tissues essential for vision. One consequence of this immunoregulation is compromised immune mediated elimination of intraocular tumors. For example, certain murine tumor cell lines which are rejected by host immune responses when transplanted in the skin grow progressively when placed in the anterior chamber (a.c.) of the eye. Progressive ocular tumor growth occurs despite induction of tumor-specific CD8+ T cell responses capable of eliminating a subsequent tumor challenge in the skin or opposite eye. Why these CD8+ T effectors fail to eliminate established ocular tumors is not known. It is well appreciated that growth of tumors in the a.c. induces the generation of immunosuppressive CD8+ T regulatory (Treg) cells. However, the contribution of CD8+ Treg in ocular tumor progression remains unclear. Several studies indicate that these CD8+ Treg target responding CD4+ T cells to inhibit their induction of macrophage-dependent delayed type hypersensitivity (DTH) responses to tumor antigens (Ags). However, induction of tumor-specific CD4+ T cell responses does not assure intraocular tumor elimination. This review is focused on how CD8+ Treg could influence the tumoricidal activity of ocular tumor-specific CD8+ T effector cells.

Inflammatory response to intravitreal injection of gold nanorods.

AIM: To evaluate the utility of gold nanorods (AuNRs) as a contrast agent for ocular optical coherence tomography (OCT). METHODS: Mice were intravitreally injected with sterile AuNRs coated with either poly(strenesulfate) (PSS-AuNRs) or anti-CD90.2 antibodies (Ab-AuNRs), and imaged using OCT. After 24 h, eyes were processed for transmission electron microscopy or rendered into single cell suspensions for flow cytometric analysis to determine absolute numbers of CD45(+) leukocytes and subsets (T cells, myeloid cells, macrophages, neutrophils). Generalised estimation equations were used to compare cell counts between groups. RESULTS: PSS-AuNRs and Ab-AuNRs were visualised in the vitreous 30 min and 24 h post-injection with OCT. At 24 h, a statistically significant increase in leukocytes, comprised primarily of neutrophils, was observed in eyes that received either AuNR in comparison to eyes that received saline. The accumulation of leukocytes was equal in eyes given PSS-AuNR or Ab-AuNR. Endotoxin-resistant C3H/HeJ mice also showed ocular inflammation after injection with AuNRs, indicating that the inflammatory response was not due to lipopolysaccharide contamination of AuNRs. CONCLUSIONS: Although AuNRs can be visualised in the eye using OCT, they can induce ocular inflammation, which limits their use as a contrast agent.

A caveat for T cell transfer studies: generation of cytotoxic anti-Thy1.2 antibodies in Thy1.1 congenic mice given Thy1.2+ tumors or T cells.

Thy1.1 congenic B6.PL mice were used to simultaneously monitor Thy1.2+ E.G7-OVA tumors transplanted in the a.c. of the eye and i.v.-transferred tumor-specific Thy1.2+ CTLs to determine mechanisms that inhibit the tumoricidal activity of CTL responses in mice with established ocular tumors. Transferred CTLs were systemically deleted in mice with established ocular tumors. However, this deletion was not a unique mechanism of immune evasion by ocular tumors. Rather, development of Thy1.2+ tumors in the eye or skin of B6.PL mice generated cytotoxic anti-Thy1.2 antibodies that eliminated a subsequent Thy1.2+ T cell transfer. Anti-Thy1.2 immune responses in B6.PL mice were influenced by the route of antigen administration, as the serum concentration of cytotoxic anti-Thy1.2 antibodies was 92-fold greater in mice with eye tumors in comparison with mice with skin tumors. In addition, anti-Thy1.2 immune responses were detected in B6.PL mice given naïve Thy1.2+ T cells i.p. but not i.v. Anti-Thy1.2 responses were augmented in B6.PL mice with ocular Thy1.2+ EL-4 tumors that did not express OVA, suggesting immunodominance of OVA antigen over Thy1.2. Thy1.1+ T cells given i.p. was not immunogenic in Thy1.2 congenic mice. These data reaffirm that the introduction of antigens in the a.c. induces robust antibody responses. Experimentation using allotypic differences in Thy1 between donor cells and recipient mice must consider cytotoxic anti-Thy1 antibody generation in the interpretation of results.

Influence of immune privilege on ocular tumor development.

Mechanisms that maintain ocular immune privilege may contribute to ocular tumor progression by inhibiting tumoricidal immune responses. Consistent with that notion are observations from transplantable tumor models in mice demonstrating that the tumoricidal activity of CD8(+) cytolytic T lymphocytes (CTL) may be inhibited directly by interfering with CTL effector function in the eye or indirectly by abrogating the effector function of CD8+ T cell-activated intratumoral macrophages that are critical for ocular tumor rejection. In addition, epigenetic gene regulation by factors within the ocular tumor environment favors the generation of tumor variants that are resistant to CD8(+) CTL. Intratumoral macrophages may be essential for eliminating these variants because, unlike CTL, their tumoricidal activity is nonspecific. Hence, the inhibition of macrophage effector function within the eye, presumably to preserve immune privilege by minimizing ocular immunopathology, may hasten the outgrowth of tumor escape variants which contributes to ocular tumor progression.

Activated CD11b+ CD15+ granulocytes increase in the blood of patients with uveal melanoma.

PURPOSE: To determine whether activated CD11b(+) CD15(+) granulocytes increase in the blood of patients with uveal melanoma. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from the blood of patients with primary choroidal/ciliochoroidal uveal melanomas (six women, four men; age range, 46-91 years) and healthy control donors (14 women, 10 men; age range, 50-81 years). The expression of CD15 and CD68 on CD11b(+) myeloid cells within PBMCs and primary uveal melanomas was evaluated by flow cytometry. CD3zeta chain expression by CD3epsilon(+) T cells in PBMCs and within primary uveal melanomas was measured as an indirect indication of T-cell function. RESULTS: The percentage of CD11b(+) cells in PBMCs of patients with uveal melanoma increased 1.8-fold in comparison to healthy donors and comprised three subsets: CD68 negative CD15(+) granulocytes, which increased 4.1-fold; CD68(-) CD15(-) cells, which increased threefold; and CD68(+) CD15(low) cells, which were unchanged. A significant (2.7-fold) reduction in CD3zeta chain expression on CD3epsilon(+) T cells, a marker of T-cell dysfunction, was observed in PBMCs of patients with uveal melanoma in comparison with healthy control subjects and correlated significantly with the percentage of CD11b(+) cells in PBMCs. CD3zeta chain expression on T cells within primary tumors was equivalent to CD3zeta expression in PBMCs of the same patient in four of five patients analyzed. CONCLUSIONS: Activated CD11b(+) CD15(+) granulocytes expand in the blood of patients with uveal melanoma and may contribute to immune evasion by ocular tumors by inhibiting T-cell function via decreasing CD3zeta chain expression.

Accumulation of immunosuppressive CD11b+ myeloid cells correlates with the failure to prevent tumor growth in the anterior chamber of the eye.

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor, E.G7-OVA, was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice, although both routes primed OVA-specific immune responses, which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice, suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA, but few were detected in primary ocular tumors. Nevertheless, growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice, and CD8(+) T cell numbers were increased within eyes, suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However, CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus, CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye.

Injection of soluble antigen into the anterior chamber of the eye induces expansion and functional unresponsiveness of antigen-specific CD8+ T cells.

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8(+) CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8(+) TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8(+) T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8(+) T cells.