<figure><img src="image_1.png" /></figure>

HAL
open science

# Expression of FcμR by bovine mononuclear blood leukocytes

Florence Gilbert, Rodrigo Prado Martins, Pascal Rainard

<figure><img src="image_2.png" /></figure>

## To cite this version:

Florence Gilbert, Rodrigo Prado Martins, Pascal Rainard. Expression of FcμR by bovine mononuclear blood leukocytes. Developmental and Comparative Immunology, 2025, 162, pp.105304. 10.1016/j.dci.2024.105304. hal-04891562

HAL Id: hal-04891562

https://hal.inrae.fr/hal-04891562v1

Submitted on 16 Jan 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_3.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_4.png" /></figure>

# Expression of FcμR by bovine mononuclear blood leukocytes

Florence B. Gilbert *, Rodrigo P. Martins, Pascal Rainard

ISP, INRAE, Université de Tours, Nouzilly, France

## ARTICLE INFO

**Keywords:**

Cattle

Fcμ receptor

IgM

Monocyte

B lymphocyte

Phagocytosis

## ABSTRACT

The receptor for IgM has been identified a few years ago, but its expression by bovine mononuclear cells has not yet been studied. We used rabbit antibodies against bovine FcμR to begin to fill this gap. Anti-FcμR antibodies bound to B lymphocytes and monocytes, although less than to neutrophils. Nonclassical and intermediate monocytes (CD172a<sup>pos</sup> CD16<sup>pos</sup>) bound nonspecifically to rabbit antibodies, complicating analysis, but they bound more anti-FcμR antibodies than control antibodies, indicating that they also express the FcμR. They also express more C5a receptors than classical monocytes. Anti-FcμR antibodies did not bind to CD3<sup>pos</sup> αβT lymphocytes (both CD4<sup>pos</sup> and CD8<sup>pos</sup>) and γδT cells. At low temperature but not at physiological temperature, purified bovine IgM bound to all monocytes and strongly to all B cells, but hardly to CD3<sup>pos</sup> T cells. Monocytes and B cells bound human IgA, but IgA did not compete, whereas unlabeled bovine IgM competed for binding of labeled IgM. This supports the role of the FcμR, and not the FαμR, in IgM binding. Finally, we showed that monocytes were able to ingest bacteria opsonized with serum deprived of IgG, indicating their ability to perform IgM-dependent phagocytosis. In conclusion, surface expression of FcμR by unstimulated blood leukocytes was demonstrated on B cells and monocytes, but not on T cells.

## 1. Introduction

IgM is a key component of humoral immunity, both systemic and mucosal. It is the first antibody produced during an immune response, a potent activator of the complement system, and an important contributor to humoral immune defense against pathogens. Some of its effector functions depend on interactions with various membrane IgM-binding receptors. On leucocytes, two receptors can bind IgM with high affinity: the FcαμR, which binds IgA and IgM, and the FcμR, which is specific for IgM (Blandino and Baumgarth, 2019). In mice and humans, the FcαμR is predominantly expressed by antigen-presenting cells, follicular dendritic cells and B lymphocytes, whereas the FcμR is mainly expressed by B lymphocytes (Kubagawa et al., 2014; Shibuya et al., 2000). After binding of IgM at physiological temperatures, the FcμR is rapidly internalized. The FcμR has been identified a few years ago and shown to be expressed by a variety of leukocytes (Kubagawa et al., 2009, 2021). Having shown that bovine neutrophils express the newly described FcμR (Gilbert and Rainard, 2024), our aim was to expand our knowledge on FcμR expression by bovine leukocytes and on the role of IgM as an opsonic antibody isotype. According to the expression of the FcμR by human and murine leucocytes, it is expected that bovine B cells express the receptor and bind IgM. In contrast, the expression of FcμR by T

lymphocytes is uncertain as human cells express the receptor but murine cells do not (Kubagawa et al., 2019). Expression by monocytes is also open to question because the FcμR would not be expressed by murine or human myeloid cells (Blandino and Baumgarth, 2019; Honjo et al., 2013). Furthermore, it has not been conclusively demonstrated that bovine monocytes bind to IgM and use them as opsonic antibodies to phagocytose pathogens (Grewal et al., 1978). In cattle, three monocytes subpopulations have been described based on the expression of CD14 and CD16, the classical monocytes (CD14<sup>pos</sup> CD16<sup>neg</sup>), the non-classical monocytes (CD14<sup>low</sup> CD16<sup>pos</sup>), and the intermediate monocytes (CD14<sup>pos</sup> CD16<sup>pos</sup>) (Corripio-Miyar et al., 2015; Elnaggar et al., 2016; Hussen et al., 2013; Talker et al., 2018). We studied FcμR expression and IgM binding in these subpopulations. We used custom-made antibodies against bovine FcμR (Gilbert and Rainard, 2024) to investigate the expression of the receptor on blood leucocytes, with neutrophils as positive control cells. We also purified bovine IgM to test whether their binding correlates with FcμR expression and allows monocytes to ingest bacteria in an IgM-dependent manner.

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: florence.gilbert@inrae.fr (F.B. Gilbert).</footnote>
## 2. Materials and methods

### 2.1. Blood sampling and peripheral blood mononuclear cells (PBMC) isolation

Jugular venous blood was collected in 10-mL K₃EDTA vacutainers (Becton Dickinson) from animals of the INRAE UE-PAO herd of Holstein cows. Samplings were performed with the approval of the “Val de Loire” Ethics Committee for Animal Experimentation and were registered to the French National Committee for Animal Experimentation under N°2016040410531486. Animal care was compliant with all applicable provisions established by the European directive 2010/63/UE.

Blood samples were first centrifuged at low speed (400 g) for 10 min at 22 °C with a swinging bucket rotor and the upper layer of plasma containing platelets was discarded. The tubes were then centrifuged at 1000 g for 20 min with the brake in the medium position. The buffy coat cells and the upper part (1/10) of the red pellet were collected in order to keep some granulocytes as FcμR-positive control cells. Red cells were lysed with ACK lysing buffer (Thermo Fisher Scientific). Afterwards, the cells were washed twice with Dulbecco's phosphate-buffered saline without Ca²⁺ and Mg²⁺ (DPBS) supplemented with 1 mg/mL bovine serum albumin (BSA; low endotoxin for cell culture, Sigma; DPBS-A), and finally adjusted in DPBS-A at 10⁷ cells/mL.

Alternatively, when assaying phagocytosis by monocytes, PBMC were prepared by centrifugation with an Optiprep™ density gradient (ProteoGenix SAS) to remove granulocytes and resuspended in RPMI1640 supplemented with 1 mg/mL BSA (RPMI-A). First, 15 ml of a 1.077 g/ml solution of Optiprep™ was centrifuged in a 50 ml Leuco-sep™ tube (Corning); then, 30 ml of blood diluted twice in DPBS was loaded into the tube and a centrifugation was performed at 700 g for 25 min at 22 °C without brake. The plasma layer was removed and the PBMC layer was recovered and transferred to a 50 ml tube, resuspended in 40 ml DPBS-A-2 mM EDTA and centrifuged at 300 g for 8 min at 22 °C. After two additional washing steps, the PBMC were finally adjusted to 10⁷ cells/mL in DPBS-A-2 mM EDTA.

### 2.2. Preparation of rabbit antibodies to bovine cell receptors

Rabbit antibodies to bovine FcμR were prepared as described (Gilbert and Rainard, 2024). Briefly, rabbit antibodies to residues 66–81 KYVREEFKHRVTLEQC from bovine FcμR (NCBI reference sequence XP_002693941.2) were affinity purified using the corresponding peptides coupled to EAH-Sepharose 4B gels (Cytiva). Antibodies to the bovine C5aR1 (CD88) were prepared by immunizing rabbits with the synthetic peptide NH2-TPDYSDYDKWTSNPDVLVDC-COOH (residues 8–26 of bovine C5aR1) coupled to keyhole limpet hemocyanin. This peptide sequence has been shown to induce antibodies recognizing the bovine C5aR1 (Nemali et al., 2008). Both peptide sequences are surface-exposed parts of the receptors. Then, antibodies were purified by affinity chromatography using the peptide as antigen. Affinity-purified rabbit antibodies to ovalbumin (anti-OVA) served as “isotypic control” for non-specific binding to bovine cells. Rabbit antibodies to ovalbumin or FcμR were labeled with Readilink™ 647/674 antibody labeling kit (Bio-Rad) as per manufacturer instructions.

### 2.3. Phenotyping of PBMC by flow cytometry

Phenotyping of T cells was carried out after the strategy described in (Tucker et al., 2023), B cells after the strategy described in (Roos et al., 2023), and monocytes after (Hussen et al., 2013), using the primary and secondary antibodies listed in Supplementary Table 1. After antibody incubations on ice and centrifugations at 4 °C, dead cells were labeled with Fixable Viability Dye eFluor 450 (eBioscience). Maintaining cells at low temperature allows measurement of IgM binding, while IgM release occurs at 37 °C (Gilbert and Rainard, 2024; Nakamura et al., 1993; Ohno et al., 1990). Labeled cells were examined using a LSR Fortessa™ X-20

(Becton-Dickinson) flow cytometer and data were analyzed with the Kaluza software (Beckman Coulter). Dead cells were excluded from the analysis and live cells gated according to their scattering and antibody labeling.

### 2.4. Purification of bovine immunoglobulin isotypes and binding to PBMC

Bovine IgM, IgG₁ and IgG₂ were prepared from a pool of “normal bovine serum” (NBS) from 10 cows as described (Gilbert and Rainard, 2024). An IgG-depleted pooled serum was prepared by passing the serum twice on a Hi-Trap Protein G column (5 mL, Cytiva) as described (Gilbert and Rainard, 2024). This preparation, designated NBS-ΔG, was stored in portions at -80 °C. It was used in the phagocytosis assay as a stable source of IgM. The IgG bound on the Protein G column were eluted and stored at -80 °C for further use as FcR blocking agent. The effective depletion of IgG (IgG₁ and IgG₂) and the presence of IgM in NBS-ΔG were verified by ELISA. Briefly, sheep antibodies directed against anti-bovine IgG(H + L) (Jackson ImmunoResearch) were distributed at 2 µg/mL in a 96 wells ELISA plate to capture the antibodies contained in the IgG-depleted serum. After an overnight incubation at 4 °C, the plate was blocked with PBS-gelatin at 0.5% (w/v) and serial dilution of the NBS-ΔG were distributed in the plate. After a 1 h incubation at 37 °C, the wells were washed and monoclonal antibodies directed against bovine IgG₁ (MCA627GA, Bio-Rad), IgG₂ (MCA2441GA, Bio-Rad) and IgM (I6137, Sigma-Merck) were distributed. After another incubation period and washing step, goat HRP-conjugated affinipure anti-mouse IgG(H + L) antibodies (Jackson ImmunoResearch) were added and finally, the reactions were developed with 3,3',5,5'-tetramethylbenzidine (TMB) and OD read at 450 nm. The values indicated that no IgG₁ and IgG₂ remained in the NBS-ΔG and that the concentration of IgM was about 2.5 mg/mL. Purified IgM were labeled with APC-Cy7 using the LYNX Rapid APC-Cy7 Antibody Conjugation Kit® from Bio-Rad. Serum human IgA were from Sigma-Aldrich. Their binding to bovine B cells and monocytes was demonstrated with F(ab')₂ fragment anti-human IgG(H + L)-PE (Supplementary Table 1).

To study the competition between human IgA and bovine IgM for FcμR binding, cells were incubated for 30 min on ice with different concentrations (0, 5, 20, 50, 100 µg/mL) of unlabeled human IgA or bovine IgM. After two washes with ice-cold buffer, labeled IgM (IgM APC-Cy7, 1 µg/mL) as well as anti-CD14 and anti-CD40 antibodies were incubated with the cells on ice for 30 min. After two washes with protein-free buffer and labeling of dead cells with FVD450, flow cytometry analysis was performed.

### 2.5. Phagocytosis assay

Phagocytic activity of monocytes was evaluated using the pHrodo™ Red Staphylococcus aureus BioParticles™ phagocytosis kit for flow cytometry (Invitrogen). PBMC, prepared with the centrifugation step with Optiprep™ to eliminate residual neutrophils, were labeled with anti-CD14-SBV790 antibody (Supplementary Table 1). Phagocytic activity of monocytes (CD14⁺ cells) was based on the fluorescence in the PE channel, as pHrodo fluoresces strongly only at acidic pH, such as in phagocytic vacuoles. Sa pHrodo™ (28 µg in 10 µL DPBS) were pre-incubated with 10 µL of either RPMI-A supplemented with 10 mM HEPES (RPMI-AH) (no opsonins), 5% NBS, 2 mg/mL IgG₁, or 5% NBS-ΔG for 15 min at 37 °C. Then, 50 µL of PBMC were added to reach a cell concentration of 2.5 x 10⁶/mL. C5a (10 nM, final concentration) was added and the phagocytic mixtures incubated under agitation for 45 min. The tubes were then placed on ice to stop phagocytosis, and the cells were analyzed by flow cytometry. Phagocytosis intensities with either no opsonins (RPMI-AH), IgG₁, or NBS-ΔG (stable source of IgM) were expressed relative to the proportion of monocytes that fluoresced in the PE channel when bacteria were opsonized with 5% NBS (set at 100).
### 3. Results and discussion

The aim of this study was to expand knowledge on the expression of FcµR by bovine leukocytes. We recently discovered that bovine neutrophils express the FcµR and that this receptor was the only receptor they use to bind IgM and phagocytose IgM-opsonized bacteria (Gilbert and Rainard, 2024). This is peculiar to the bovine because the FcµR has been reported to be expressed essentially by lymphocytes (Kubagawa et al., 2017). This prompted us to examine FcµR expression by blood leucocytes, limiting our investigation to surface expression as a first step.

We used affinity-purified rabbit anti-ovalbumin antibodies to study the non-specific binding of rabbit IgG by bovine blood leukocytes by flow cytometry. A small population of cells with low side scatter bound anti-OVA antibodies. This binding was reduced but not canceled by the addition of bovine IgG (5 mg/mL) in an attempt to block FcγRs (Supplementary Fig. 1). We investigated which IgG receptor could be responsible for this nonspecific binding. The strongest binding to anti-OVA antibodies was by the monocytes (CD172a<sup>pos</sup>) expressing the FcγRIII (CD16), i.e., non-classical monocytes (Supplementary Fig. 1A). In this experiment, we used 20 µg/mL of anti-OVA antibodies, a rather high concentration, because this was the concentration of anti-FcµR which gave the best results with neutrophils (Gilbert and Rainard, 2024). In order to prove that the anti-FcµR antibody specifically binds to FcµR (rather than non-specifically binding to the cells) the concentration of the anti-FcµR antibody should be similar to that of the anti-OVA antibody. We tested if a lower concentration would be useable even with CD16<sup>pos</sup> cells. At a lower concentration (2.5 µg/mL), rabbit anti-OVA antibodies slightly bound to monocytes (CD172a<sup>pos</sup>), whereas anti-C5aR antibodies at the same concentration (2.5 µg/mL) bound strongly only to the nonclassical monocytes (CD172A<sup>pos</sup> CD16<sup>pos</sup>) (Supplementary Fig. 1B). At this concentration, anti-C5aR antibodies bound strongly to 100% of bovine neutrophils (not shown). However, high concentrations (10–20 µg/mL) of anti-FcµR antibodies were necessary to show receptor expression by mononuclear leucocytes, and unfortunately at these concentrations the “isotype control” anti-OVA antibodies highly bound to CD16<sup>pos</sup> cells. This prevented us from drawing a definitive conclusion regarding the expression of the FcµR by NKp46<sup>pos</sup> cells which highly express CD16, although they bound slightly more antibodies to FcµR (Suppl. Fig. 2C).

As a first approach to study FcµR expression by blood leucocytes, we used antibodies to CD14 and CD40 to identify monocytes (CD40<sup>pos</sup> CD14<sup>pos</sup>) and B lymphocytes (CD40<sup>pos</sup> CD14<sup>neg</sup>) (Roos et al., 2023) (Fig. 1A). Rabbit antibodies to FcµR coupled to AF647 bound to CD40<sup>pos</sup> cells better than did rabbit anti-OVA control antibodies (Fig. 1B). Two subpopulations of CD40<sup>pos</sup> cells could be distinguished (Fig. 1C). A small CD40<sup>pos</sup> cell subpopulation which bound markedly both anti-OVA and anti-FcµR antibodies looked like the intermediate (CD172a<sup>high</sup>, CD14<sup>high</sup>) and non-classical (CD172a<sup>high</sup>, CD14<sup>low</sup>) monocytes (Fig. 1D and E). This speculation is in line with the strong non-specific binding of rabbit antibodies by CD14<sup>low</sup> CD16<sup>pos</sup> non-classical monocytes (Supplementary Fig. 1). Demonstrating expression by B cells was rather straightforward, as these cells do not bind too strongly to rabbit immunoglobulins (Fig. 1E). Accounting for the non-specific labeling of cells with anti-OVA antibodies, it appeared that B lymphocytes and monocytes, classical and non-classical, express the FcµR, although less than neutrophils (Fig. 1F).

Then we investigated the binding of anti-FcµR antibodies to bovine T lymphocytes. Surface expression of FcµR was measured on CD3<sup>pos</sup> T lymphocytes, using anti-FcµR antibodies and anti-OVA as control of nonspecific binding of rabbit antibodies. The gating strategy used allowed us to distinguish CD4<sup>pos</sup>, CD8<sup>pos</sup>, and Tγδ CD3<sup>pos</sup> cells (Fig. 2A). None of these populations bound the anti-FcµR better than the anti-OVA antibodies (Fig. 2B). There was some variation in the binding of the anti-OVA antibodies, the Tγδ lymphocytes showing the highest affinity. By comparison, neutrophils were less labeled with anti-OVA antibodies,

whereas they showed a strong fluorescence with the anti-FcµR antibodies (Fig. 2B). There was little variation between the cows tested for anti-OVA or anti-FcµR binding, except for the neutrophils with the anti-FcµR (Fig. 2C). In conclusion, these results indicate that none of the analyzed T cell populations surface-expressed the FcµR.

We then investigated whether FcµR expression corresponded to the ability to bind IgM, using custom-labeled bovine IgM (IgM-APC-Cy7) to study IgM binding to blood leukocytes. We identified B lymphocytes and monocytes based on CD14 and CD40 expression, and we restrained the identification of T lymphocytes to CD3 expression as they did not bind antibodies to FcµR (Supplementary Fig. 2A). Increasing concentration of labeled IgM bound increasingly to monocytes and B lymphocytes (Fig. 2D). The binding of IgM-APC-Cy7 (20 µg/mL) was marked and showed some variability when tested on the cells of four cows (Supplementary Fig. 2B). Binding to T lymphocytes (CD3<sup>pos</sup>) was very low or negative (Supplementary Fig. 2B). To test whether IgM could bind to the FcµR, we studied the binding of IgA to monocytes and B lymphocytes and the capacity of IgA or IgM to compete with the binding of labeled IgM. Both monocytes and B lymphocytes bound human IgA (Supplementary Fig. 2C). Human IgA have been shown to bind to bovine monocytes, and this was interpreted as a demonstration that bovine monocytes express an IgA-binding FcR (Zhang et al., 1995). As to bovine B lymphocytes, we have not found previously published data showing their binding to IgA (Kacskovics, 2004), but binding of IgA to human B lymphocytes has been documented (Lydyard and Fanger, 1982). The identity of the receptor remains to be established. Bovine IgM, but not human IgA, competed with the binding of labeled IgM (Supplementary Fig. 2D), which excludes binding of IgM by the FcαµR. These results support the view that monocytes and B lymphocytes, but not CD3<sup>pos</sup> lymphocytes, express the IgM FcµR receptor. Expression of FcµR by bovine B cells was expected as murine and human B cells have long been known to bind IgM, and have been found to express the FcµR (Kubagawa et al., 2009; Sanders et al., 1987; Shima et al., 2010). The case of monocytes was more complicated. An intriguing issue was the non-specific binding of rabbit antibodies to a monocyte subpopulation, which resisted blockade with bovine IgG. We identified the subpopulation as intermediate and non-classical monocytes: the cells strongly labeled with anti-OVA antibodies were CD172a<sup>pos</sup> CD16<sup>pos</sup> monocytes (Supplementary Fig. 1A). We suspect that the non-specific binding of rabbit antibodies is mediated by CD16 (FcγRIII), which is rather unexpected as it is a low affinity receptor for IgG, binding only to aggregated IgG or immune complexes (Noble et al., 2023). Maybe aggregates in our affinity-purified antibodies were the culprit. Homogeneous binding of IgM (Fig. 2D) by all monocytes suggests that non-classical monocytes do not express more FcµR than do classical monocytes. If intermediate and non-classical monocytes expressed FcµR more than classical monocytes, a subpopulation binding more labeled IgM would have been detected, which was not the case.

Finally, we investigated the capacity of monocytes to use IgM as opsonic antibodies. A preliminary experiment showed that, as with neutrophils (Gilbert and Rainard, 2024), C5a was necessary for the IgM-dependent phagocytosis but not for the opsonization with NBS, probably because NBS contains low amounts of C5a (Rainard et al., 1998). When PBMC were incubated with pHrodo™ red Sa bioparticles, CD14<sup>pos</sup> cells ingested only the bioparticles opsonized with NBS or NBS-ΔG, a stable source of IgM (Supplementary Fig. 3A). This result was obtained consistently with the cells of four cows (Supplementary Fig. 3B). Purified IgG<sub>1</sub> did not allow monocytes to ingest the bioparticles (Supplementary Fig. 3B), consistent with previous results showing that freshly isolated bovine monocytes poorly ingest IgG<sub>1</sub>-opsonized targets (Ngaira et al., 1983; Noble et al., 2023). This brings new information on bovine monocytes, as their capacity to use IgM had not been settled by previous studies (Grewal et al., 1978; Ngaira et al., 1983; Rossi and Kiesel, 1977). Our results support IgM-dependent phagocytosis by bovine monocytes.

Incidentally, we showed that non-classical monocytes express high
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Binding of anti-FcμR antibodies to B lymphocytes and monocytes. A) gating strategy to identify live monocytes and B cells based on expression of CD14 and CD40. B) CD40<sup>pos</sup> cells (monocytes and B cells) bind anti-FcμR better than anti-OVA antibodies, with a subpopulation showing stronger binding of rabbit antibodies. C) Binding of rabbit AF647-linked antibodies to OVA (grey trace) or FcμR (red trace) to CD40<sup>pos</sup> cells. D) Binding of anti-OVA antibodies to CD40<sup>pos</sup> CD172a<sup>pos</sup> cells (monocytes). Highly positive OVA-AF647 cells are CD172a<sup>high</sup> CD14<sup>medium</sup> (intermediate and non-classical monocytes, green dots). E) Binding of anti-FcμR-AF647 antibodies to monocytes and B cells. Intermediate and non-classical monocytes (green dots) show the highest binding. F) Comparison of the binding of anti-OVA (OVA CD40<sup>pos</sup>) and anti-FcμR to neutrophils, B cells, and monocytes.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Expression of the FcμR by blood T lymphocytes. A) Gating strategy to distinguish viable CD3<sup>pos</sup> T cells. B) Binding of anti-OVA (negative control, grey histograms) or anti-FcμR antibodies to CD4<sup>pos</sup>, CD8<sup>pos</sup>, and γδ T cells. Binding to neutrophils in the same experiment is shown as a positive control. Results representative of one experiment with 6 cows. C) Variation in binding of anti-OVA antibodies (3 cows) or anti-FcμR antibodies (6 cows) to T cells, with neutrophils as control. Black dots are for MFI with anti-OVA and red dots for MFI with anti-FcμR antibodies. D) Binding of increasing concentrations (0, 1, 5, 20, or 40 µg/mL) of labeled IgM to monocytes or B lymphocytes.</figcaption></figure>

levels of C5aR, contrary to classical monocytes (Supplementary Fig. 1). As a low concentration of specific antibody was sufficient, non-specific binding of rabbit antibodies was not an issue. This is consistent with previous finding showing that most bovine monocytes react poorly with

the monoclonal anti-C5aR antibody 3D5, based on the same C5aR sequence as our rabbit antibodies (Nemali et al., 2008). High expression of CD16 and CD172a distinguishes the non-classical monocytes (Hussen et al., 2013), to which we can add the expression of C5aR1 (CD88). The
functional activities of bovine non-classical monocytes is still a matter of speculations. Bovine non-classical monocytes showed reduced phagocytosis capacity and were considered non-inflammatory (Hussen et al., 2013). Gene transcription analysis supported anti-inflammatory, anti-viral and tissue repair functions of non-classical monocytes (Talker et al., 2022). However, non-classical monocytes were shown to be more endocytic than classical monocytes (Corripio-Miyar et al., 2015). The expression of CD16 and FcµR by non-classical monocytes but a reduced phagocytic capacity may seem paradoxical. Furthermore, the expression of C5aR suggests that they could migrate to inflammatory sites. As suggested, functional testing will be necessary to clarify the issue (Hussen and Schuberth, 2017).

In conclusion, our study shows that bovine B cells and monocytes express FcµR, whereas circulating T cells do not. Complementing our previous study that showed that bovine neutrophils express FcµR (Gilbert and Rainard, 2024), these results confirm that the IgM receptor expression pattern is species specific, as previously suggested (Honjo et al., 2013). Its broad pattern of expression suggests that it plays a variety of roles in the immune system, consistent with the major role the IgM antibody isotype plays in the immune defense in the bovine species (Sun et al., 2020). Much remains to be done to fill the many gaps in our knowledge on this subject, such as refining and completing FcµR cellular expression pattern and exploring its activities. Because of its potential importance, genetic determinism and individual variability of expression or activity of the FcµR deserve to be evaluated. This could open up new ways to increase cattle resistance to disease.

### CRediT authorship contribution statement

**Florence B. Gilbert:** Writing – review & editing, Resources, Investigation. **Rodrigo P. Martins:** Writing – review & editing, Investigation. **Pascal Rainard:** Writing – original draft, Supervision, Investigation, Conceptualization.

### Funding

This work was supported by INRAE and the EGER program of APIS-GENE (grant name MASTICELLS).

### Acknowledgments

The competent technical assistance from the UE-PAO dairy farm staff in blood sample collection and animal care is greatly appreciated.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.dci.2024.105304.

### Data availability

Data will be made available on request.

### References

* Blandino, R., Baumgarth, N., 2019. Secreted IgM: new tricks for an old molecule. J. Leukoc. Biol. 106, 1021–1034.

* Corripio-Miyar, Y., Hope, J., McInnes, C.J., Wattegedera, S.R., Jensen, K., Pang, Y., Entrican, G., Glass, E.J., 2015. Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity. Vet. Res. 46, 112.

* Elnaggar, M.M., Abdellrazeq, G.S., Mack, V., Fry, L.M., Davis, W.C., Park, K.T., 2016. Characterization and use of new monoclonal antibodies to CD11c, CD14, and CD163

to analyze the phenotypic complexity of monocyte subsets. Vet. Immunol. Immunopathol. 178, 57–63.

* Gilbert, F.B., Rainard, P., 2024. Expression of the receptor for IgM (FcµR) by bovine neutrophils. Dev. Comp. Immunol. 160, 105235.

* Grewal, A.S., Rouse, B.T., Babiuk, L.A., 1978. Characterization of surface receptors on bovine leukocytes. Int. Arch. Allergy Appl. Immunol. 56, 289–300.

* Honjo, K., Kubagawa, Y., Kubagawa, H., 2013. Is Toso/IgM Fc receptor (FcmuR) expressed by innate immune cells? Proc. Natl. Acad. Sci. U. S. A. 110, E2540–E2541.

* Hussen, J., Duvel, A., Sandra, O., Smith, D., Sheldon, I.M., Zieger, P., Schuberth, H.J., 2013. Phenotypic and functional heterogeneity of bovine blood monocytes. PLoS One 8, e71502.

* Hussen, J., Schuberth, H.J., 2017. Heterogeneity of bovine peripheral blood monocytes. Front. Immunol. 8, 1875.

* Kacskovics, I., 2004. Fc receptors in livestock species. Vet. Immunol. Immunopathol. 102, 351–362.

* Kubagawa, H., Honjo, K., Ohkura, N., Sakaguchi, S., Radbruch, A., Melchers, F., Jani, P. K., 2019. Functional roles of the IgM Fc receptor in the immune system. Front. Immunol. 10, 945.

* Kubagawa, H., Oka, S., Kubagawa, Y., Torii, I., Takayama, E., Kang, D.W., Gartland, G.L., Bertoli, L.F., Mori, H., Takatsu, H., Kitamura, T., Ohno, H., Wang, J.Y., 2009. Identity of the elusive IgM Fc receptor (FcmuR) in humans. J. Exp. Med. 206, 2779–2793.

* Kubagawa, H., Oka, S., Kubagawa, Y., Torii, I., Takayama, E., Kang, D.W., Jones, D., Nishida, N., Miyawaki, T., Bertoli, L.F., Sanders, S.K., Honjo, K., 2014. The long elusive IgM Fc receptor, FcmuR. J. Clin. Immunol. 34 (Suppl. 1), S35–S45.

* Kubagawa, H., Skopnik, C.M., Al-Qaisi, K., Calvert, R.A., Honjo, K., Kubagawa, Y., Teuber, R., Aliabadi, P.M., Enghard, P., Radbruch, A., Sutton, B.J., 2021. Differences between human and mouse IgM Fc eceptor (FcmicroR). Int. J. Mol. Sci. 22.

* Kubagawa, H., Skopnik, C.M., Zimmermann, J., Durek, P., Chang, H.D., Yoo, E., Bertoli, L.F., Honjo, K., Radbruch, A., 2017. Authentic IgM Fc receptor (FcmuR). Curr. Top. Microbiol. Immunol. 408, 25–45.

* Lydyard, P.M., Fanger, M.W., 1982. Characteristics and function of Fc receptors on human lymphocytes. Immunology 47, 1–17.

* Nakamura, T., Kubagawa, H., Ohno, T., Cooper, M.D., 1993. Characterization of an IgM Fc-binding receptor on human T cells. J. Immunol. 151, 6933–6941.

* Nemali, S., Siemsen, D.W., Nelson, L.K., Bunger, P.L., Faulkner, C.L., Rainard, P., Gauss, K.A., Jutila, M.A., Quinn, M.T., 2008. Molecular analysis of the bovine anaphylatoxin C5a receptor. J. Leukoc. Biol. 84, 537–549.

* Ngaira, J.M., Nantulya, V.M., Musoke, A.J., Hirumi, K., 1983. Phagocytosis of antibody-sensitized Trypanosoma brucei in vitro by bovine peripheral blood monocytes. Immunology 49, 393–400.

* Noble, A., Paudyal, B., Schwartz, J.C., Mwangi, W., Munir, D., Tchilian, E., Hammond, J. A., Graham, S.P., 2023. Distinct effector functions mediated by Fc regions of bovine IgG subclasses and their interaction with Fc gamma receptors. Front. Immunol. 14, 1286903.

* Ohno, T., Kubagawa, H., Sanders, S.K., Cooper, M.D., 1990. Biochemical nature of an Fc mu receptor on human B-lineage cells. J. Exp. Med. 172, 1165–1175.

* Rainard, P., Sarradin, P., Paape, M.J., Poutrel, B., 1998. Quantification of C5a/C5a (desArg) in bovine plasma, serum and milk. Vet. Res. 29, 73–88.

* Roos, E.O., Bonnet-Di Placido, M., Mwangi, W.N., Moffat, K., Fry, L.M., Waters, R., Hammond, J.A., 2023. OMIP-085: cattle B-cell phenotyping by an 8-color panel. Cytometry Part A: the journal of the International Society for Analytical Cytology 103, 12–15.

* Rossi, C.R., Kiesel, G.K., 1977. Bovine peripheral blood monocyte cultures: growth characteristics and cellular receptors for immunoglobulin G and complement. Am. J. Vet. Res. 38, 559–563.

* Sanders, S.K., Kubagawa, H., Suzuki, T., Butler, J.L., Cooper, M.D., 1987. IgM binding protein expressed by activated B cells. J. Immunol. 139, 188–193.

* Shibuya, A., Sakamoto, N., Shimizu, Y., Shibuya, K., Osawa, M., Hiroyama, T., Eyre, H.J., Sutherland, G.R., Endo, Y., Fujita, T., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Phillips, J.H., Lanier, L.L., Nakauchi, H., 2000. Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes. Nat. Immunol. 1, 441–446.

* Shima, H., Takatsu, H., Fukuda, S., Ohmae, M., Hase, K., Kubagawa, H., Wang, J.Y., Ohno, H., 2010. Identification of TOSO/FAIM3 as an Fc receptor for IgM. Int. Immunol. 22, 149–156.

* Sun, Y., Huang, T., Hammarstrom, L., Zhao, Y.F., 2020. The immunoglobulins: new insights, Implications and applications. In: Lewin, H.A., Roberts, R.M. (Eds.), Annual Rev. Animal Biosci. 8, 145–169, 2020.

* Talker, S.C., Barut, G.T., Lischer, H.E.L., Rufener, R., von Munchow, L., Bruggmann, R., Summerfield, A., 2022. Monocyte biology conserved across species: functional insights from cattle. Front. Immunol. 13.

* Talker, S.C., Baumann, A., Barut, G.T., Keller, I., Bruggmann, R., Summerfield, A., 2018. Precise delineation and transcriptional characterization of bovine blood dendritic-cell and monocyte subsets. Front. Immunol. 9.

* Tucker, N., Cunha, P., Gilbert, F.B., Rambault, M., Santos, K.R., Remot, A., Germon, P., Rainard, P., Martins, R.P., 2023. Bovine blood and milk T-cell subsets in distinct states of activation and differentiation during subclinical *Staphylococcus aureus* mastitis. J. Reprod. Immunol. 156, 103826.

* Zhang, G., Young, J.R., Tregaskes, C.A., Sopp, P., Howard, C.J., 1995. Identification of a novel class of mammalian Fc gamma receptor. J. Immunol. 155, 1534–1541.