Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.

Introduction Mice BLT1-deficient (BLT1-KO: Ltb4r1 -/-) mice were generated as described previously [53,54] and backcrossed with BALB/c or C57BL/6 mice for more than 12 generations. Wild-type (WT) mice (BALB/c) were purchased from Japan SLC (Shizuoka, Japan) or Kyudo (Saga, Japan). All mice were maintained in a filtered-air laminar-flow enclosure in a specific pathogen-free facility and given standard laboratory food and water. All mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).

Ethics statement
All animal experiments were approved by the Ethical Committee for Animal Experiments in Kyushu University and Juntendo University. All the studies in this manuscript were carried out in accordance with approved guidelines and regulations.

Plasmids
The pCXN2 vector [55] was used for the expression of various GPCRs. Constructs encoding various untagged or N-terminally FLAG-tagged GPCRs were constructed in-house as previously described [56].

Antibody purification and labeling
Abs were affinity-purified from the culture supernatant using Protein G Sepharose (GE Healthcare) according to the manufacturer's protocol. Affinity-purified mAbs were separated by SDS-PAGE on 10% acrylamide gels and stained with Coomassie brilliant blue. The concentration of purified Ig was determined by UV absorbance at 280 nm. The isotype of the 7A8 mAb was determined using a mouse mAb isotype kit (Hycult biotechnology). Biotinylated 7A8 mAb (7A8-Biotin) or AF488-labeled mAb (7A8-AF488) were prepared using sulfosuccinimidobiotin (Thermo Fisher Scientific) or AF488 carboxylic acid, tetrafluorophenyl ester, bis (triethylammonium salt) (Thermo Fisher Scientific) according to the manufacturer's protocol, respectively.

Depletion assay
WT mice (BALB/c, female, 8-10 weeks old) were i.p. injected with 100 μg of 7A8, 1A8 (anti-Ly6G mAb; Bio X Cell, West Lebanon, NH), or anti-Gr-1 mAbs, or PBS (as a control). PBL were collected at 1 day after mAb injection as described above (see "Flow cytometry"), and were then stained with mAbs and analyzed by flow cytometry.

Statistics
Data are expressed as the mean ± SEM. ANOVA tests were used for multiple comparisons. P-values <0.05 were considered statistically significant. All statistical analyses were performed using Prism, version 5.0 (GraphPad Software, La Jolla, CA).

Results and discussion
Establishment of a hybridoma cell line that produces an anti-mouse BLT1 monoclonal antibody To establish a hybridoma that produces a high-affinity mAb for mBLT1, BLT1-WT and BLT1-KO mice were immunized with L1.2-mBLT1 cells that stably expressed untagged mBLT1 (Fig 1A). After eight i.p. injections of L1.2-mBLT1 cells, mice received four additional immunizations with the same cells plus MPL/TDM adjuvant. Plasma from BLT1-KO mice, but not BLT1-WT mice, was found to be immunoreactive to BTL1 (Fig 1B). Approximately 1,200 hybridoma clones were generated by fusing SP2/0 myeloma cells with splenocytes and lymph node cells from immunized BLT1-KO mice. Culture supernatant from one clone, 7A8, positively stained CHO-mBLT1 cells by flow cytometry. Immunoglobulin was purified from the 7A8 culture supernatant using Protein G Sepharose, and the purity and recovery of 7A8 mAb was analyzed by SDS-PAGE (Fig 1C). The isotype of the 7A8 mAb was determined to be IgG 1 . The average yield of functional 7A8 mAb was approximately 2.3 mg from 100 ml of culture supernatant.

The 7A8 mAb recognizes the second extracellular loop of mBLT1
To determine the epitope of mBLT1 recognized by 7A8, four peptides with sequences derived from the extracellular domains of mBLT1 (an N-terminal region and three harboring loops) (Fig 3A) were synthesized and used in a competition assay with 7A8. As shown in Fig 3B, the binding reactivity of 7A8 was clearly decreased in the presence of a peptide comprising amino acids 159-190 of mBLT1 (mBLT1  ), which is located in the second extracellular loop. Next, the binding of 7A8 to mBLT1  was directly examined by SPR (Fig 3C). The 7A8 mAb bound directly to mBLT1  with an association (k a ) and dissociation rate constant (k d ) of 1.05 × 10 4 M -1 s -1 and 6.49 × 10 −3 s -1 , respectively. The equilibrium dissociation

Detection of endogenous mouse BLT1 with 7A8
The ability of the 7A8 mAb to detect endogenous mBLT1 was analyzed next. 7A8-Biotin was prepared and used for flow cytometry. As shown in Fig 4A, 7A8-Biotin positively stained Gr-1-positive (Gr-1 + ) granulocytes, CD11b + granulocytes/monocytes, and F4/80 + monocytes in the peripheral blood from naïve BLT1-WT mice. No 7A8 staining of naïve CD4 + T cells, CD8 + T cells, and B220 + B cells were observed, consistent with the previously reported lack of BLT1 expression on these cells [16]. Binding of 7A8 to Gr-1 + granulocytes was not observed in BLT1-KO mice but was not affected by BLT2 deficiency (data not shown), confirming the specificity of the 7A8 mAb. Both CD115 + (a canonical monocyte marker) and Ly6C + (a classical or pro-inflammatory monocyte marker) monocytes expressed BLT1 (data not shown). Further analysis showed that BLT1 was expressed in both CCR2-high (CCR2 hi ) inflammatory and CCR2-low (CCR2 lo ) resident monocytes (Fig 4B). Furthermore, BLT1 expression was clearly detected only in a subset of CD4 + T cells, Th1 cells differentiated in vitro, but was not obvious in Th0 and Th2 cells (Fig 4C). Taken together, these data indicate that 7A8 is sensitive and specific enough to detect endogenous mBLT1.

Validation of 7A8 for immunohistochemistry of mBLT1
In general, it is difficult to stain mouse tissues using monoclonal or polyclonal Abs derived from mouse due to high background staining caused by endogenous mouse IgG and mouse Fc receptors expressed on immune cells such as B cells, macrophages, and DC. We therefore tried to detect mBLT1-expressing cells in the mouse spleen with 7A8 using a tyramide signal amplification system. The 7A8 mAb was able to detect Gr-1 + granulocytes in the red pulp (RP) and Gr-1 + monocytic cells (such as monocytes, macrophages, or DC) in the white pulp (WP) of BLT1-WT mice, but 7A8 staining was not observed in BLT1-KO mice (Fig 5). These data clearly show that 7A8 mAb is a highly sensitive and specific mAb for mBLT1.

Clone 7A8 can deplete granulocytes and monocytes from the peripheral blood
To examine whether 7A8 can be used for depletion of mBLT1-expressing cells in vivo, 7A8, 1A8, or anti-Gr-1 mAbs was administered to WT mice (100 μg/mice, i.p.). PBL were collected and analyzed by flow cytometry at 1 day after mAb injection. As shown in Fig 6A, Gr-1 hi CD11b hi (C-CR2 -F4/80 -) granulocytes and F4/80 lo CD11b-middle (CD11b mid ) (CCR2 lo Gr-1 mid ) monocytes were depleted in peripheral blood in either cases; however, CCR2 hi CD11b hi (F4/80 lo Gr-1 hi ) inflammatory monocytes were eliminated only by injection of the 7A8 mAb. The 7A8 administration significantly depleted granulocytes and monocytes (Fig 6B). Administration of 1A8 and anti-Gr-1 mAb depleted most of the granulocytes with some depleting effects on monocyte used to stain CHO-mBLT1 cells and mock transfectants. Data were analyzed by one-way ANOVA, followed by the Bonferroni post-hoc test: ***, p < 0.001. (C) Affinity and kinetic measurements for 7A8 mAb by Biacore analysis. The four peptides were flowed over 7A8 immobilized onto a sensor chip, and the binding interactions were measured in resonance units (RU). https://doi.org/10.1371/journal.pone.0185133.g003 Establishment of a novel anti-mouse BLT1 mAb, 7A8  PBL (A, B) and in vitro differentiated CD4 + T cell subsets (C) with 7A8. (A) PBL were stained with 7A8-Biotin and several lineage markers, and staining was analyzed separately in Gr-1 + granulocytes, CD11b + granulocytes/monocytes, F4/80 + monocytes, CD4 + T cells, CD8 + T cells, and B220 + B cells from naïve BLT1-WT (black outlines) and BLT1-KO (gray filled histograms) mice. (B) Monocytes were stained with 7A8-Biotin and anti-CCR2 mAb from the peripheral blood of naïve WT mice. The mIgG 1 -Biotin and rIgG 2b mAb were used as isotype controls. (C) Th0, Th1 and Th2 cells were stained with 7A8-AF488 (black outlines) or mIgG-AF488 (gray filled histograms) as a control. https://doi.org/10.1371/journal.pone.0185133.g004 Establishment of a novel anti-mouse BLT1 mAb, 7A8 subsets. Thus, the 7A8 mAb will be useful to analyze the physiological and pathophysiological roles of BLT1 + granulocytes and monocytes in vivo.
In this study, we successfully established an anti-mBLT1 mAb (clone 7A8) with high specificity and sensitivity for exogenous (Fig 2) and endogenous (Figs 4 and 5) mBLT1 by immunizing BLT1-KO mice with BLT1-overexpressing cells (Fig 1). Endogenous BLT1 is abundantly expressed in granulocytes, which have a very short life span in vivo. Therefore, WT mice develop peripheral immunological tolerance for BLT1, necessitating the use of BTL1-KO mice for Ab generation. Although several mAbs can be obtained by immunization with a general Establishment of a novel anti-mouse BLT1 mAb, 7A8 antigen (e.g., recombinant fusion protein or plasmid DNA) using gene-deficient mice [58,59,60,61,62,63], little has been reported on anti-GPCR mAb generation by this strategy. Thus, this approach might be useful for generating highly specific and sensitive mAbs against other GPCRs and orphan GPCRs. We confirmed that 7A8 can be used for detection of mBLT1 on tissue by immunohistochemistry (Fig 5). In addition, 7A8 significantly depleted BLT1 + granulocytes and monocytes (Fig 6), but not lymphocytes (data not shown), from the peripheral blood. Furthermore, 7A8 bound the second extracellular loop of mBLT1 (Fig 3), and did not affect the LTB 4 binding and intracellular signaling (S1 Fig). These results are consistent with previous studies showing that the transmembrane domains III, V and VI of BLT1 are involved Establishment of a novel anti-mouse BLT1 mAb, 7A8 in the LTB 4 binding [64,65,66]. Our data suggests that 7A8 mAb will be a very useful tool to clarify the physiological and pathophysiological functions of BLT1 in various disease models by quickly eliminating BLT1-expressing cells without inducing the compensatory gene expression caused by BLT1 deficiency. Future studies will examine the effects of depleting BLT1-expressing cells on the onset and progression of several inflammatory diseases in animal models.
We also observed that BLT1 was expressed in circulating CCR2 hi inflammatory monocytes and CCR2 lo resident monocytes in the peripheral blood (Fig 4B). The physiological and pathophysiological roles of BLT1 in those subsets are unknown. In general, inflammatory (or classical) monocytes (CCR2 hi Ly6C hi CX 3 CR1 lo ) are recruited to inflamed tissues, where they produce inflammatory cytokines (e.g., IL-1beta and TNF-alpha) in response to infection or tissue damage. They become differentiated into marcophages or DC during acute inflammation and chronic inflammation. In the steady state or during homeostatic inflammation, they become tissue-resident macrophages, which readily engulf dying or dead cells (including apoptotic or necrotic cells) and cellular debris, contributing to the maintenance of tissue homeostasis. Tissue-resident (or non-classical) monocytes (CCR2 lo Ly6C lo CX 3 CR1 hi ) patrol locally to clear cellular debris. These cells can also produce antiinflammatory cytokines (e.g., IL-10), although their cellular function is not fully understood [67,68,69]. Recent reports show that both monocyte subsets (classical and non-classical) can polarize into alternatively activated (or M2-type) macrophages [70,71,72], although the molecular mechanisms of this conversion remain poorly understood. Future studies in our group will investigate the functions of BLT1 in both classical and non-classical monocytes using the 7A8 mAb.

S1 Fig. 7A8 mAb does not affect interaction of LTB 4 and mBLT1. (A)
Competitive binding assay of 7A8 mAb with a radioactive ligand. Microsomal fractions were mixed with [ 3 H]LTB 4 , and added with or without 7A8 (Total). A non-specific binding (NS) was determined with 2,000-fold concentration of non-labeled LTB 4 in the same preparation (n = 2-3 for Total, n = 2 for NS). Data were analyzed by one-way ANOVA, followed by the Newman-Keuls posthoc test: ns, not significant. (B) The effect of 7A8 on LTB 4 -BLT1 signaling. L1.2-mBLT1, L1.2-FLAG-mBLT1 cells or mock transfectants were incubated with 7A8 mAb, and calcium mobilization was measured by stimulation with LTB 4 (n = 2-3). RFI: relative fluorescent intensity. Data were analyzed by two-way ANOVA: ns, not significant. (TIF)