Macrophage inflammatory protein-1alpha as a costimulatory signal for mast cell-mediated immediate hypersensitivity reactions

Abstract

Regulation of the immune response requires the cooperation of multiple signals in the activation of effector cells. For example, T cells require signals emanating from both the TCR for antigen (upon recognition of MHC/antigenic peptide) and receptors for costimulatory molecules (e.g., CD80 and CD60) for full activation. Here we show that IgE-mediated reactions in the conjunctiva also require multiple signals. Immediate hypersensitivity reactions in the conjunctiva were inhibited in mice deficient in macrophage inflammatory protein-1alpha (MIP-1alpha) despite normal numbers of tissue mast cells and no decrease in the levels of allergen-specific IgE. Treatment of sensitized animals with neutralizing antibodies with specificity for MIP-1alpha also inhibited hypersensitivity in the conjunctiva. In both cases (MIP-1alpha deficiency and antibody treatment), the degranulation of mast cells in situ was affected. In vitro sensitization assays showed that MIP-1alpha is indeed required for optimal mast cell degranulation, along with cross-linking of the high-affinity IgE receptor, FcepsilonRI. The data indicate that MIP-1alpha constitutes an important second signal for mast cell degranulation in the conjunctiva in vivo and consequently for acute-phase disease. Antagonizing the interaction of MIP-1alpha with its receptor CC chemokine receptor 1 (CCR1) or signal transduction from CCR1 may therefore prove to be effective as an antiinflammatory therapy on the ocular surface.

Figure 1

Location of MIP-1α–expressing cells in the allergen-challenged conjunctiva. (A) Localization of MIP-1α mRNA-positive cells in the allergen-challenged conjunctiva. Slides were exposed for 4 days, developed, and counterstained with H&E staining. (B) Schematic description of conjunctival mast cell distribution. Note colocalization with MIP-1α–positive cell distribution as shown in A. (C) MIP-1α mRNA-positive cells were also shown in the tarsal conjunctival area of allergen-challenged mice (3 weeks exposure). (D) MIP-1α–positive cells in PBS-challenged conjunctiva (3 weeks exposure). Ep, conjunctival epithelium; M, meibomian gland. Magnification, ×40 (A), ×200 (C and D).

Figure 2

Conjunctival mast cells express CCR1 and respond to recombinant MIP-1α. (A) Conjunctival mast cells from a naive mouse stain positive for CCR1. (B) Toluidine blue staining of mast cells in a serial section. CCR1⁺ mast cells are indicated with asterisks in A. (C) Subconjunctival injection of recombinant MIP-1α results in the chemotaxis of mast cells into the conjunctiva in naive mice. Statistically significant elevation of total mast cell numbers in MIP-1α–injected conjunctiva compared with eotaxin-1– or sham-injected controls (4 h after injection). MIP-1α increased degranulated mast cell count proportionately. n = 6 per group; P < 0.05. Values are expressed as mean ± SEM. Eot, eotaxin-1.

Figure 3

Impairment of allergen-induced immediate hypersensitivity reaction and mast cell degranulation in MIP-1α–deficient mice. (A) Immediate hypersensitivity reaction, shown here as % maximal clinical score (defined as sum of each clinical symptom scores), was abolished in MIP-1α–deficient (–/–) mice. n = 13 per group; P < 0.05. (B) Degranulation of mast cells was significantly impaired in MIP-1α–deficient mice. (C) Total mast cell counts in these mice were not affected by MIP-1α deficiency. Values are expressed as mean ± SEM.

Figure 4

Local chemokine production and allergen-specific Ig synthesis in wild-type and MIP-1α–deficient mice. (A) Profiles of chemokine induction in MIP-1α–deficient eye homogenates by RNase protection assay. MIP-1α deficiency did not affect induction of eotaxin-1, MIP-2, MCP-1, or IFN-γ–inducible protein 10 (IP-10) 24 hours after allergen challenge. Each lane represents RNAs isolated from 2 representative eyes in each group. Ltn, lymphotactin. (B) Allergen-specific serum Ig levels in immunized mice. MIP-1α deficiency did not impair the synthesis of serum Igs (IgE, IgG1, IgG2a). n = 9 per group. Levels of allergen-specific Igs (IgE, IgG1, IgG2a) in the mock-immunized mice were below detection limits (data not shown). Values are expressed as mean ± SEM.

Figure 5

Clinical scores are reduced and immediate sensitivity reactions impaired in mast cell–deficient W/W^v mice. (A) Clinical scores were significantly suppressed in W/W^v mice as compared with mast cell–competent WT mice (n = 20 per group; P < 0.05). (B) Each clinical symptom, including tearing, conjunctival redness, lid edema, and conjunctival edema was also suppressed in W/W^v mice. (C) Evans blue dye extravasation 90 minutes after allergen challenge was assessed in W/W^v and WT mice. W/W^v mice show minimal dye extravasation, while WT mice showed a significant increase in extravasation in response to allergen challenge (n = 8 per group; P < 0.05). Values are expressed as mean ± SEM.

Figure 6

In vivo neutralization of MIP-1α inhibits mast cell activation and clinical symptoms in the allergen-challenged conjunctiva. (A) Suppression of allergen-induced immediate hypersensitivity by anti–MIP-1α antibody treatment. Mice primed for immediate hypersensitivity reaction were administered anti–MIP-1α monoclonal antibody (30 μg/injection) intravenously 1 hour before allergen challenge. Clinical scores assessed on day 3 were significantly reduced (n = 12 per group; P < 0.05). (B) Each clinical symptom, including conjunctival edema, lid edema, conjunctival redness, and tearing, was reduced by antibody treatment. (C) The frequency of degranulated mast cells following allergen challenge was also significantly reduced (n = 12 per group; P < 0.05). (D) Late-phase recruitment of mast cells was also assessed in WT mice and following MIP-1α blockade at 24 hours after challenge. Mast cell recruitment was significantly suppressed by antibody treatment (n = 12 per group; P < 0.05). (E) An analysis of mast cell degranulation and clinical scores showed a positive correlation between these 2 indices. (F) Kinetics of inhibitory effect of MIP-1α antibody treatment on clinical scores (100 μg/injection). Clinical scores were significantly suppressed on days 1, 2, and 3 (P < 0.05). Values are expressed as mean ± SEM.

Figure 7

Augmentation of allergen-induced histamine release by MIP-1α. The experimental design involved a passive sensitization assay using isolated conjunctival tissue from naive mice. The upper panel shows histamine release from the in vitro–sensitized tissue challenged with eotaxin-1 (100 ng/ml), MIP-1α (100 ng/ml), or compound 48/80 (1 mg/ml) without HSA-DNP (allergen) addition. The lower panel shows histamine release from the in vitro–sensitized tissue challenged with HSA-DNP in media, eotaxin-1 (100 ng/ml), or MIP-1α (100 ng/ml). MIP-1α supplementation significantly augmented allergen–induced (HSA-DNP–induced) histamine release of in vitro–sensitized conjunctival mast cells, while eotaxin-1 or vehicle had no effect. Compound 48/80 solution served as a positive control of mast cell degranulation. Values are expressed as mean ± SEM.