Allergy Asthma Immunol Res.  2017 Jul;9(4):360-367. 10.4168/aair.2017.9.4.360.

CCR3 Monoclonal Antibody Inhibits Eosinophilic Inflammation and Mucosal Injury in a Mouse Model of Eosinophilic Gastroenteritis

• Song DJ
• Shim MH
• Lee N
• Yoo Y
• Choung JT

• KMID: 2378199
• DOI: http://doi.org/10.4168/aair.2017.9.4.360

Abstract

PURPOSE
Although the role of eosinophils in eosinophilic gastroenteritis (EGE) is not fully understood, they are believed to be a principal effector cell. Previous studies have demonstrated that eotaxin and its specific receptor, cysteine-cysteine chemokine receptor-3 (CCR3), play a central role in eosinophil trafficking into the gastrointestinal (GI) tract. Thus, we examined the targeting of CCR3 as a potential therapeutic intervention for EGE in a mouse model.
METHODS
Eight- to 10-week-old BALB/c mice were intraperitoneally sensitized and intragastrically challenged with ovalbumin (OVA). Different groups of mice were administered either an anti-CCR3 antibody or a control IgG by intraperitoneal injection 1 hour before each OVA challenge. Eosinophilic inflammation in the intestinal mucosa, mucosal injury, and severity of diarrhea were compared between different groups at 1 hour after final OVA challenge.
RESULTS
Anti-CCR3 antibody reduced the number of eosinophils in peripheral blood and intestinal mucosa, but not in bone marrow. This reduction was associated with restoration of reduced villous crypt ratio, increased intestinal epithelial cell proliferation, and weight loss induced by OVA challenge. However, Anti-CCR3 antibody had no effect on the level of OVA specific immunoglobulin E (IgE) and the expression of critical chemokines or cytokines in eosinophil trafficking into the GI tract, such as eotaxin-1, interleukin (IL)-5, and IL-13.
CONCLUSIONS
Anti-CCR3 antibody significantly reduced the severity of eosinophilic inflammation, mucosal injury, and diarrhea in a mouse model of food allergen-induced GI eosinophilic inflammation. CCR3 may be a novel therapeutic target for treatment of EGE and other GI eosinophil-mediated diseases.

Keyword

CCR3; eosinophilic gastroenteritis; eosinophils

MeSH Terms

Animals
Bone Marrow
Chemokine CCL11
Chemokines
Cytokines
Diarrhea
Eosinophils*
Epithelial Cells
Gastroenteritis*
Gastrointestinal Tract
Immunoglobulin E
Immunoglobulin G
Immunoglobulins
Inflammation*
Injections, Intraperitoneal
Interleukin-13
Interleukins
Intestinal Mucosa
Mice*
Ovalbumin
Ovum
Weight Loss
Chemokine CCL11
Chemokines
Cytokines
Immunoglobulin E
Immunoglobulin G
Immunoglobulins
Interleukin-13
Interleukins
Ovalbumin

Figure

• 

  Fig. 1 Experimental protocol. Mice were intraperitoneally sensitized on days 0 and 14, and intragastrically challenged with OVA on days 28, 30, 32, 35, 37, and 39. One hour before each OVA challenge, anti-CCR3 or isotype control antibody was intraperitoneally administered (arrows). Mice were sacrificed 1 hour after the final OVA challenge, and their jejunums were analyzed. OVA, ovalbumin; CCR3, cysteine-cysteine chemokine receptor-3.

• 

  Fig. 2 Eosinophils in the bone marrow and PB. PB was collected by cardiac puncture and bone marrow cells were flushed from the femurs on day 39. Cell suspensions were cytospun onto microscope slides and then stained with Wright-Giemsa. Differential cell counts were performed in PB (A) and BM (B) cells. Data are expressed as means±SE. PB, peripheral blood; BM, bone marrow; SE, standard error. *P<0.05.

• 

  Fig. 3 Eosinophils and mast cells in the jejunum. Eosinophils and mast cells in the jejunum were detected using anti-MBP immunostain (A) and chloroacetate esterase staining (B), respectively. Red dots indicate MBP+ or chloroacetate esterase+ cells. The numbers of eosinophils (C) and mast cells (D) per mm2 of jejunal lamina propria were quantitated in different groups of mice. Data are expressed as means±SE. MBP, major basic protein; SE, standard error. *P<0.001.

• 

  Fig. 4 Expression of eotaxin, IL-5, IL-13, and serum OVA-pecific IgE. Intestinal tissue was homogenized in 2.0 mL of PBS (pH 7.4) and supernatants assayed by ELISA for eotaxin-1 (A), IL-5 (B), and IL-13 (C). Intestinal tissue protein levels were quantitated using a BCA protein assay (Pierce, Rockford, IL, USA). Results are expressed as pg/mg intestinal mucosa protein. Serum OVA-specific IgE levels (D) were measured by ELISA on day 39 in different groups of mice. Data are expressed as means±SE. IL, interleukin; OVA, ovalbumin; IgE, immunoglobulin E; PBS, phosphate buffered saline; ELISA, enzyme-linked immunosorbent assay; SE, standard error. *P<0.05; †P<0.005.

• 

  Fig. 5 Mucosal epithelial cell proliferation in the jejunum. The incorporation of BrdU in the mucosal epithelial cells was measured 3 hours after intraperitoneal injection of 5′-BrdU on day 39. (A) BrdU-labeled epithelial cells (dark brown dots) in the jejunal mucosa. (B) The percentage of BrdU-labeled cells per 500 crypt cells was quantitated in different groups of mice. Data are expressed as means±SE. BrdU, bromodeoxyuridine; SE, standard error. *P<0.05; †P<0.001.

• 

  Fig. 6 Villus/crypt morphologic changes. Using a light microscope attached to an image-analysis system, villous height and crypt depth in jejunum were measured in ten randomly selected crypt-villous units from hematoxylin-eosin stained sections at ×10 magnification. Data are expressed as means±SE. SE, standard error. *P<0.001.

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