Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy

Abstract

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

Keywords: Rab44; differentiation of blood cells; granulocytes; immune responses; nickel allergy.

Figure 1

Upregulation of Rab44 in macrophage differentiation. THP-1 cells were induced to M0 macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) (100 nM) at 37 °C for 24 h. M0 macrophages were differentiated into M1 macrophages by incubation with lipopolysaccharide (LPS) (0.1 mg/mL) and interferon (IFN)-γ (20 ng/mL), or M2 macrophages by incubation with IL-4 (20 ng/mL) and IL-13 (20 ng/mL) at 37 °C for 0–24 h. (a) Representative images of M1- and M2-type macrophages are shown. Bars: 20 μm. (b) Quantitative RT-PCR determination of Rab44 mRNA expression in M1- or M2-type macrophages for the indicated times. Data are represented as the mean ± S.E. of values from three independent experiments.

Figure 2

Different immune responses between wild-type (WT) and Rab44-knockout mice (KO) to LPS administration. (a,b) WT and Rab44-KO female mice were intraperitoneally injected with 0.2 mg of LPS (O55:B5). After the injection, blood was collected from the mandibular or tail vein at 12 h. The cytokine levels of TNF-α (a) and IL-10 (b) in the sera were measured using ELISA. (c,d) After 48 h of injection, the blood was analyzed with a hematology counter, Thika (ARKRAY) (WT, n = 8; KO, n = 5). * p < 0.05 compared between WT and KO mice. Granulocytes (c) and lymphocytes (d) counts (left panels), and percentages in white blood cells (right panels).

Figure 3

Sensitivity of macrophages to nickel solution. Bone marrow-derived macrophages derived from wild-type (WT) and Rab44-knockout (KO) mice were incubated with the indicated concentrations of Ni₂SO₄ solution for 24 h. Viability was determined using a Cell Counting Kit. * p < 0.05 compared to WT mice.

Figure 4

Responses of mice fed with nickel-water. (a) Gross appearance of wild-type (WT) and Rab44-knockout (KO) mice fed with nickel-water for 2 months. (b) Serum IL-1β level of WT and Rab44-KO mice fed with nickel-water for 2 months. (c) Granulocytes counts and percentage in white blood cells of WT and Rab44-KO mice before or after administration of nickel-water. (d) Monocyte percentage in white blood cells of WT and Rab44-KO mice fed with nickel-water for 2 months. * p < 0.05 and ** p < 0.01 compared between WT and KO mice.

Figure 5

Ear swelling of mice in response to nickel solution. Wild-type (WT) and Rab44-knockout (KO) mice were sensitized with LPS and a nickel solution. Ten days after sensitization, the mice were fed with nickel-water for 2 months. Two months later, the mice were injected with the nickel solution. Ear thickness was measured before the stimulation and for 72 h after the stimulation. * p < 0.05 compared to WT.

Figure 6

Immunofluorescence analysis of CD11b and Gr-1-positive cells in nickel-induced ear in wild-type (WT) and Rab44-knockout (KO) mice. (a) The fixed sections of the nickel-treated ear were blocked with 1.0% bovine serum albumin in PBS. The samples were incubated with rabbit anti-CD11b IgG (1:400) and rat anti-Gr-1 IgG (1:200), as the primary antibody, followed by fluorescent labelling with Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 555-conjugated anti-rat IgG, and then observed by microscopy. Bars: 50 μm. White arrowheads indicate the antibody reactive cells. (b,c) Quantitative analysis of CD11b and Gr-1-positive cells per μm² after visualization by microscopy. Data are represented as the mean ± S.E. of values from three independent experiments. Asterisks indicate statistical significance compared to the control; * p < 0.05.