An intracameral injection of antigen induces in situ chemokines and cytokines required for the generation of circulating immunoregulatory monocytes

Abstract

Anterior Chamber-Associated Immune Deviation (ACAID) induced by an intracameral injection of antigen generates antigen-specific regulatory splenic T cells that suppress specifically cell-mediated immunity specific for the injected antigen. Circulating F4/80(+) cells recovered from mice receiving an intracameral injection of antigen are thought to be ocular in origin and induce the development of thymic and splenic regulatory T cells. We have shown previously that after the intracameral injection of antigen there is a CCR2/CCL2-dependent infiltration of circulating F4/80(+) cells into the anterior chamber associated with the generation of circulating, ACAID-inducing F4/80(+) monocytes. Here we tested the hypothesis that the intracameral injection of antigen induces events in the anterior chamber that are associated with the induction of circulating immunoregulatory monocytes that induce the suppression of cell-mediated immunity. The intracameral injection of antigen resulted in aqueous humor (i) a time- dependent increase of CCL2 and CCL7, (ii) a transient increase in TNF-α, and (iii) an infiltration of CD11b(hi), Gr1(hi) and F4/80(+) as well as F4/80(-) and Gr1(hi) peripheral blood cells into the anterior chamber. Further characterization of these F4/80(+) cells revealed that they are Ly 6C(hi), LY6G(lo) or negative, 7/4 (LY6B)(hi), CD115(+), CD45(+), CD49B(+), and CD62 L(+). Antibody-mediated neutralization of TGF-β in situ in the anterior chamber prevented the induction of circulating, ACAID-inducing monocytes and ACAID. These cells did not increase in the irides of ACAID-refractory CCR2-/- and CCL2-/- mice that received an intracameral injection of antigen. Our results extend our suggestion that ACAID is initiated as the result of a mild proinflammatory response to intracameral injection that results in the infiltration of a CCR2(+) subset of monocytes into the anterior chamber where there is a TGF-β-dependent induction of an immunosuppressive phenotype in the infiltrated monocytes that recirculate to induce antigen-specific regulatory T cells.

Figure 1. CCL2, CCL7 and TNF-α levels in the aqueous humor after intracameral injection.

Aqueous humor was collected at different time intervals following the intracameral injection of OVA. CCL2, CCL7 and TNF-α levels in aqueous humor were detected by ELISA and are expressed as pg/ml. The data were pooled from 4 independent experiments for CCL2, 2 independent experiments for CCL7 and TNF-α and 2 independent experiments for MCP-3. A minimum of 6 replicates were used for each experiment.

Figure 2. Intracameral (AC) injection results in infiltration of, F4/80⁺, CD11b^hi Gr1^hi monocytes and this process is greatly reduced in CCR2 or CCL2 null animals.

Sixteen hours post intracameral injection of OVA irides were recovered from 10 eyes. A single cell suspension of the pooled irides was prepared and the cells were stained for specific monocyte markers as described in material and methods and compared the same with naive group or with cells from irides of either CCR2 null or CCL2 null animals treated identically. (A) Initial gating on the scatter plot is done as shown. A comparison between naïve iris monocytes and iris- gated monocytes recovered from mice receiving an intracameral injection of antigen (AC) stained with anti-CD11b, F4/80 and anti- Gr1. Only the group receiving an intracameral injection contains cells that are CD11b^hi, F480⁺and Gr1^hi. CD11b^hi but F4/80⁻ cells are also observed in mice receiving an intracameral injection of antigen. The figure is representative of 8 experiments (P<0.001). (B) The composite histogram shows that CD11b^hi population in AC groups but not in naïve group. The CD11b^hi F4/80⁺ Gr1^hi cells are increased in AC wild type group but only marginally increased in CCL2^−/− group after AC injection. (C) The bar diagrams shows the F/480⁺ cell population present in the wild type and CCL−/− mice receiving an intracameral injection as cells/10⁶ acquired. The figures are representative of 2 experiments. (D) AC injection results in neutrophil infiltration. Cells are gated on a population of higher SSC and intermediate FSC which in AC injection group show a Ly6G^hi peak. This population is absent in naive iris. Ly6G cells are F4/80 negative and Ly6C intermediate. (E) Comparison of 2 populations of cells in the AC injected iris group, based on their scatter properties. Ly6G hi cells are abundant in the R1 region but also are present in R2 region. These cells are also CD11b^hi.

Figure 3. F4/80 and CD11b⁺ cells infiltrating the iris after the intracameral injection of antigen also express Ly6C, 7/4 (Ly 6B) and CD45.

Preparations of naïve iris cells and cells recovered after an intracameral injection of antigen were stained with anti- F4/80 and (A) Ly6C and 7/4 (B) show a characteristic CD45 peak C) Ly6G^lo or negative, CD115, CD49b⁺, CD62L^lo. The figures are representative of 2 experiments. All the isotype controls in the histograms are shown as shaded.

Figure 4. Regulation of the increase in TNF-α and CCL2 in the anterior chamber after an intracameral injection.

Aqueous humor was collected at at 3 and 6 hour intervals following the intracameral injection of OVA. CCL2 and TNF-α levels in the aqueous humor were measured by ELISA. (A) Neutralization of TNF-α prevents the early rise of CCL2. Mice received an intracameral injection of OVA +/– anti-TNF-α antibody or isotype control. Neutralization of TNF-α via anti-TNF-α antibody at the time of AC injection significantly (P<0.05) reduced the early (3 hr) rise of (CCL2) MCP-1 but not at 6 hours. The data were pooled from 2 independent experiments and a minimum of 6 replicates were used in each group for each experiment. (B) Neutralization of TGF-β via a neutralizing antibody or TGF-β receptor kinase inhibitor (SB-431542) results in a significant reduction(P<0.05) in the rise of TNF-α induced by an intracameral injection of OVA. The data were pooled from 2 independent experiments. A minimum of 6 replicates were used for each experiment. (C) Neutralization of TGF-β via anti-TGFβ neutralizing antibody, Soluble TGF-β receptor or by TGF-β receptor kinase inhibitor (SB-431542) does not influence the CCL2 levels in the aqueous humor.(D) Neutralization of TGF-β by intracameral injection of anti-TGF-β with OVA does not block the infiltration of monocytes into the anterior chamber. Irides were removed 16 hr after intracameral injection and cell suspensions stained for Gr1 and CÎ11b. Experiments were repeated 3X.

Figure 5. Blockade of TGF-β via (A) neutralizing antibody, (B) TGF-β soluble receptor, C) TGF-βreceptor kinase at the time of the intracameral injection of OVA on the development of DTH suppression.

Intracameral injection of OVA included either vehicle only or isotype antibody control (AC positive control) or various TGF-β blocking treatments. The DTH measurements were performed 7 days after these groups were immunized with OVA. Each experiment, 5 mice/group, was conducted 3X.

Figure 6. Intracameral injection of PI3 Kinase inhibitors along with the antigen prevent the development of ACAID.

Mice received an intracameral injection of OVA or OVA +wortmannin. One week later the mice were immunized with OVA/CFA and one week later footpads were challenged with OVA. Footpad swelling was measured 24 hr after challenge and were compared between immunized mice, naïve mice and those that received an intracameral injection of vehicle + OVA with that of wortmannin and OVA. The experiments were repeated 3X.

Figure 7. AC- PBMCs from Anti-TGF-β-treated animals do not transfer DTH suppression to OVA however intravenous injection of AC- PBMCs to those animals receiving anti-TGF-β+ OVA intracameral injections rescues the suppression of DTH.

(A) Anti-TGF-β treatment at the time of intracameral injection of antigen blocks the ability of AC-PBMCs to induce the suppression of DTH. Twenty-four hr after intracameral injection of OVA and anti-TGF-β, PBMCs were recovered and 1×10⁶ recovered cells were injected IV into the naïve mice. These recipient mice were subsequently immunized with OVA/CFA. Seven days after immunizing, footpads of the mice were challenged subcutaneously and the relative increase in footpad swelling was compared between the immunized control mice and naïve mice. Each group in the final DTH measurement had 5 mice. The experiment was repeated 3X, (B) Animals receiving intracameral injections of anti-TGF-β +OVA were injected IV at 24 hours with AC-PBMCs recovered from animals receiving intracameral injections of OVA only. The recipient mice sere then immunized with OVA/CFA. Seven days after immunizing DTH measurements were done as described above. The experiment was repeated twice.