Open Access

Laminin Isoforms in Human Dental Pulp: Lymphatic Vessels Express Laminin-332, and Schwann Cell–Associated Laminin-211 Modulates CD163 Expression of M2-like Macrophages

Nagako Yoshiba, Naoki Edanami, Naoto Ohkura, Tomoki Maekawa, Naoki Takahashi, Takahiro Tsuzuno, Takeyasu Maeda, Koichi Tabeta, Kenji Izumi, Yuichiro Noiri and Kunihiko Yoshiba
ImmunoHorizons December 1, 2021, 5 (12) 1008-1020; DOI: https://doi.org/10.4049/immunohorizons.2100110

Abstract

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/β/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3β3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2–coated plates upregulated CD163 expression, compared with laminin-511/E8-α5–coated plates. Integrin α3– and integrin α6–neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin–laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

Introduction

Macrophages, immune system cells with indispensable roles in tissue-specific innate defense and organ homeostasis, can be classified into two groups: classically activated macrophages (proinflammatory; M1) and alternatively activated macrophages (anti-inflammatory and pro-healing; M2) (1). M1-like macrophages are typically induced in response to tissue damage or pathogen exposure by mediators such as IFN-γ, TNF, and LPS (2). In contrast, M2-like macrophages are a heterogeneous population of macrophages induced by various anti-inflammatory mediators, including IL-4, IL-13, IL-10, TGF-β, and M-CSF (3); these macrophages are characterized by the expression of several receptors. Mannose receptor CD206 has been consistently used as a marker of macrophages differentiated by exposure to both IL-4 and IL-10 (3); scavenger receptor CD163 is increased by exposure to IL-10 but not IL-4 (4). Tissue-resident macrophages perform immune sentinel and homeostatic functions (5), and CD163 is detected on most subsets of tissue-resident macrophages (6). During bacterial infection, CD163 on tissue-resident macrophages binds to bacteria and acts as an innate immune sensor that detects bacterial infections (7).

Dental pulp is a highly innervated, soft connective tissue encased in dentin. Nerves enter the pulp through the apical foramen and branch to form an extensive plexus of nerve fibers below odontoblasts, which are located at the periphery of the pulp and are directly associated with dentinogenesis. Schwann cells are a major cellular component of peripheral nerves and support peripheral neurons, which are critical for responses to axon damage and regeneration (8). Currently, distinct subsets of resident tissue macrophages, which are associated with peripheral nerves, are receiving increasing attention (9). We recently reported the colocalization of CD163+ macrophages with Schwann cells in healthy and carious human dental pulp, as well as during wound healing; our findings suggested that CD163 expression was modified by Schwann cells in vitro (10).

The basement membrane, a widely distributed extracellular matrix, lies at the basal aspect of epithelial and endothelial cells in addition to the surrounding muscle, adipose tissues, and Schwann cells (11). Laminins are major basement membrane proteins composed of three different chains, termed α, β, and γ. Laminin α (α1–5)-chains form heterotrimers with β (β1–3)- and γ (γ1–3)-chains, each with two to five isoforms; at least 16 laminin isoforms have been identified in mammals (12). The chain composition reflects the isoform nomenclature (13). For example, α2β1γ1 laminin is termed laminin-211. These isoforms have tissue- and development-specific distribution patterns (12). Laminin-111 and laminin-511 are expressed in mammalian embryos; laminin-511 maintains tissue homeostasis throughout life. Laminins containing α4 and α3 are present in blood vessel walls and under various epithelia, respectively. Laminins containing α2 are present in skeletal muscle and peripheral nerves. Laminin isoforms modulate cell phenotype and differentiation patterns in a tissue-specific manner (14). Recently, endothelial laminin-511 was reported to promote monocyte differentiation to macrophages (15).

Currently, the effects of laminin isoforms on macrophage phenotypes are poorly understood. On the basis of previous findings that CD163+ macrophages were associated with Schwann cells, we investigated the effects of Schwann cell–associated laminins on M2-like macrophages. We initially analyzed the gene expression and distribution patterns of laminin isoforms in human dental pulp and then assessed whether Schwann cell–associated laminins were involved in modulating macrophage phenotypes in vitro.

Materials and Methods

This research project was approved by the Niigata University Ethics Committee (2019-0049). Human healthy permanent teeth and small pieces of gingival flap from individuals aged 20–25 y, as well as third molars with caries from individuals aged 20–28 y, were obtained during the orthodontic treatment of patients who provided informed consent to be included in the study. One tooth per patient was used, and the teeth used in the current study are summarized in Table I.

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Table I.

Intact and carious teeth examined in the current study

Immunofluorescence and immunohistochemistry

The Ab that recognizes laminin α3 has limited applications when using paraformaldehyde-fixed tissues; therefore, acetone was used to fix tissues. The teeth were split open immediately after extraction under local anesthesia, and the pulp tissue was carefully removed. Cryostat sections were cut at a thickness of 10 µm, then fixed in acetone. For paraformaldehyde fixation, the extracted teeth were fixed with 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4), demineralized in 10% EDTA (pH 7.4), and then sectioned at a thickness of 10 µm. Immunostaining was performed as previously described (16), using the primary Abs listed in Table II. The secondary Abs were HRP-labeled swine anti-rabbit IgG (Dako, Glostrup, Denmark), Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 546 goat anti-mouse IgG (Invitrogen, Carlsbad, CA). The tissue sections were then examined under a Nikon E-800 fluorescence microscope equipped with a digital microscope camera (DP-80, Olympus, Tokyo, Japan). Quantification of macrophage subsets was performed as follows: five representative sections were selected from each experimental tooth and counted in 10 fields of view in similar areas per section at ×400 magnification; this quantification was performed using cellSens Standard imaging software (Olympus). Negative control staining was performed by replacing the primary Abs with PBS. Control sections did not show any specific immunoreactivity.

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Table II.

Abs used in the current study

Quantitative RT-PCR

Total RNA was extracted from dental pulp or cultured THP-1 macrophages using TRIzol reagent (Life Technologies, Carlsbad, CA), in accordance with the manufacturer’s instructions. The yield was determined by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). PrimeScript RT master mix (Takara Bio, Shiga, Japan) was used to synthesize cDNA. Quantitative real-time PCR (qRT-PCR) was performed using a StepOnePlus real-time PCR system (Thermo Fisher Scientific) with gene-specific primers and TB Green Premix Ex Taq II (Takara Bio), in accordance with the manufacturer’s instructions. To examine the expression levels and their changes, the values were normalized according to the expression of β-actin mRNA, a housekeeping gene. Primer sequences are listed in Table III.

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Table III.

Primer sequences for qRT-PCR analysis

Cell culture

THP-1 cells were obtained from RIKEN (Tsukuba, Japan). Culture medium for all experiments was DMEM (Wako, Osaka, Japan) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 10 IU/ml penicillin, and 10 µg/ml streptomycin (Wako). Twelve-well tissue culture plates (Corning Life Sciences, Corning, NY) were coated with laminin-111, -211, or -511 (BioLamina, Sundbyberg, Sweden) at 1.25 µg/cm2 in PBS. Coating with recombinant laminin E8 fragments of α2 (E8-α2) or α5 (E8-α5) (iMatrix, Matrixome, Osaka, Japan) was performed at a density of 1.25 µg/cm2. To induce the differentiation of THP-1 cells to a macrophage phenotype, they were seeded at 0.5 × 106 cells/well with culture media containing 5 ng/ml PMA. A low dose of PMA was used because it is representative of human macrophage responses and allows for enhanced responsiveness to weaker stimuli (17, 18). After 2 d, THP-1 macrophages were washed and incubated in culture media alone for 1 d. Then, they were untreated or stimulated with IL-4 (20 ng/ml, R&D Systems, Minneapolis, MN) or IL-10 (20 ng/ml, R&D Systems) for 2 d. Neutralizing Abs specific for laminin-related integrin receptors (α6 and α3) or isotype control Abs were added to the culture medium at 5 µg/ml or 10 µg/ml. The Abs used are listed in Table II. Cultures were incubated at 37°C in a humidified atmosphere with 5% CO2. Phase contrast images of cells were captured under a Nikon Eclipse Ts2 inverted microscope.

Flow cytometric analysis

A thermosensitive cell culture dish (Cepallet, DIC, Tokyo, Japan) was used for flow cytometric analysis, in accordance with the manufacturer’s instructions. Cultured macrophages were detached by replacing culture medium with cold medium, without enzyme treatment. Isolated single cells were fixed with 4% paraformaldehyde in PBS, followed by washing, and Fc receptors were blocked using an anti-CD16/32 Ab (Invitrogen). Samples were incubated with Abs against CD163 as listed in Table II. Isotype control Abs were purchased from eBioscience (San Diego, CA, USA). The labeled cells were washed twice and analyzed (NovoCyte flow cytometer; ACEA Biosciences, San Diego, CA).

Results

Identification of laminin chain expression in human dental pulp

A schematic illustration shows the well-innervated dental pulp, particularly below the odontoblasts located at the periphery of the pulp (Fig. 1A). Integrins are transmembrane receptors composed of α and β subunits; they transduce various intracellular signals (11). E8 domains of the globular domains in laminin α-chains (illustrated in (Fig. 1B) are prerequisites for integrin binding to laminins; therefore, we analyzed mRNA laminin α-chain expression patterns in healthy dental pulp. Laminin α1-chain was not expressed in any samples (Fig. 1C). Laminin α2-, α3-, α4-, and α5-chains were expressed at the same levels in the dental pulp (Fig. 1C). Laminin α2 and glial fibrillary acidic protein, a marker of Schwann cells (19), were colocalized on the longitudinal and cross-sections (Fig. 1D, upper lane), including the plexus of nerves in the peripheral area (Fig. 1D, lower lane). Unexpectedly, we detected thin-walled capillaries labeled with laminin α3; therefore, we performed double immunofluorescence staining with lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1), a marker of lymphatic vessels, and laminin α3. Surprisingly, we detected the colocalization of LYVE1 and laminin α3 on vessel structures in the middle of the dental pulp (Fig. 1Ea–c). The lumen, ∼50 µm in diameter, was connected to several irregular-shaped lumens (Fig. 1Ea–c). The peripheral regions contained double-positive vessels (Fig. 1Ed–f). Additionally, LYVE1 single-positive cells were present (Fig. 1Ea–f). Immunohistochemical staining demonstrated a vessel structure with LYVE1 (Fig. 1Eg), as well as thin-walled capillaries labeled with laminin α3 in the peripheral area (Fig. 1Eh–j). Laminin α3+ cells were positive for CD31 (Fig. 1Ek–m), a pan-endothelial marker, but they were not associated with α-smooth muscle actin (αSMA)–expressing cells composed of mural cells or pericytes of blood vessels (Fig. 1En); they were also negative for laminin α5 (Fig. 1Eo). Furthermore, laminin α3-chains were colocalized with laminin γ2-chains (Fig. 1Ep–r). Taken together, the vessels labeled with laminin α3 were considered to be pulpal lymphatic vessels; the type of laminin was identified as laminin-332 (previously named laminin-5). Laminin α4 (Fig. 1F) and α5 (Fig. 1G) immunoreactions were exclusively found in blood vessels. Human gingival basement membrane, used as negative or positive control tissue, was positive for laminin α3 and α5; it was negative for laminin α2 and α4 (Fig. 1H). The muscle in the gingival flap exhibited laminin α2 immunoreactivity (Fig. 1H).

FIGURE 1.
FIGURE 1.

Identification of laminin-α chain expression in human dental pulp.

(A) Schematic illustration of a tooth. The dental pulp is a highly innervated, soft connective tissue encased in dentin. An extensive plexus of nerve fibers forms below odontoblasts located at the periphery of the pulp. (B) Illustration of a laminin heterotrimer. G, globular. (C) mRNA expression levels of laminin α-chains in healthy dental pulp. Results are shown as means ± SEM (n = 4). Statistical analysis was performed by one-way ANOVA and Bonferroni tests. ns, not significant. (D) Double immunofluorescence staining of laminin α2-chains and glial fibrillary acidic protein (GFAP), a marker of Schwann cells, in longitudinal and cross-sections (upper panels) and in the plexus of nerves (lower panels; closer views of the boxed area are shown in the three right panels). (E) Double immunofluorescence staining of lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1), a marker of lymphatic vessels, and laminin α3-chains (Ea–Ef). Asterisks indicate the lymphatic lumens and outlined arrowheads indicate the junctions between the lumens (Ea–Ec) detected in the middle of the dental pulp. White arrows indicate double-positive cells (Ed–Ef), and arrowheads indicate single-positive cells (Ea–Ef). Immunohistochemical staining of LYVE1 (Eg) and laminin α3-chains (Eh–Ej) are shown. The arrow indicates an LYVE1-positive (brown) lymphatic vessel (Eg). Thin-walled capillaries are labeled with laminin α3 (brown) (Eh, arrows). (Ei and Ej) Closer views of the boxed area in (Eh). Double immunofluorescence staining is shown of laminin α3 and CD31 (Ek–Em), α-smooth muscle actin (αSMA) (En), or laminin α5-chains (LAMA5) (Eo) in the peripheral area of healthy human dental pulp. White arrows indicate double-positive cells, and arrowheads indicate single-positive cells (Ek–Eo). Double immunofluorescence staining of laminin α3- and laminin γ2-chains (LAMC2) (Ep–Er) is shown; arrows indicate their colocalization (Ep–Er). (F) Double immunofluorescence staining of laminin α2- and α4-chains. (G) Double immunofluorescence staining of laminin α2- and α5-chains in cross-sections and longitudinal sections. Arrowheads indicate the positive reactions of laminin α5 detected in a cross-section. (H) Immunofluorescence staining of laminin α2-, α3-, α4-, and α5-chains in the gingiva. Arrowheads indicate the gingival basement membrane. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm.

Localization of CD206+ or CD163+ cells in the human dental pulp of intact and carious teeth

We used the CD206 and CD163 markers to assess the localization of M2-like macrophages. CD206 is a well-established marker of M2-like macrophages both in vivo and in vitro, but its expression is not macrophage restricted—it is also expressed by immature dendritic cells and certain other cell types (20). Therefore, the pan-macrophage marker CD68 was used in addition to CD206. In intact teeth, elongated CD206+ cells were detected along with Schwann cells (Fig. 2Aa–c). Some oval-shaped CD206+ cells were also present (Fig. 2Ad). Double immunofluorescence staining indicated that elongated CD206+ cells were positive for CD68; oval or round-shaped CD206+ cells were positive or negative for CD68, both inside and outside of blood vessels (Fig. 2Ae–g′). Most CD163+ cells in the healthy dental pulp were also positive for CD206 (Fig. 2B). However, CD206+CD163− macrophages were present along blood vessels (Fig. 2B, inset). CD206+ cells were spread along blood vessel walls stained with laminin α5 (Fig. 2C). In contrast, CD163+ cells were localized to Schwann cell–associated laminin α2-chains (Fig. 2D). CD206+CD163+ double-positive macrophages had an elongated shape and were predominantly distributed along nerve fibers (Fig. 2D). We previously reported that CD163+ cells were colocalized with Schwann cells in carious teeth (10). Therefore, we analyzed the localization of CD206/CD163 cells in carious teeth. Strongly stained CD206+ cells with an oval or round shape were abundant under active carious lesions (Fig. 2Ea, b), with both CD68+ and CD68− findings (Fig. 2Ec, c′); they were negative for CD163 (Fig. 2F). Furthermore, most CD163+ cells were negative for CD206 (Fig. 2F). Most CD163+ cells had an elongated shape and were recognizable with Schwann cell–associated laminin α2-chains in carious teeth (Fig. 2G). Under slowly progressing carious lesions, CD206+CD163+ double-positive macrophages were observed more frequently than were CD206−CD163+ single-positive macrophages (Fig. 2H). We quantified the subsets of macrophages by examining the ratio of CD206+CD163+ double-positive macrophages to CD206−CD163+ single-positive macrophages (Fig. 2I). The ratio was significantly lower in active lesions than in intact or slowly progressing carious teeth (Fig. 2I).

FIGURE 2.
FIGURE 2.

Localization of CD206+ or CD163+ cells in human dental pulp.

(A–H) Healthy teeth (A–D) and representative images of active (E–G) or slowly progressing (H) carious teeth. (A) Immunohistochemical staining of CD206 (Aa–Ad), and double immunofluorescence staining of CD206 (Ae–Ag) and the corresponding CD68 findings (Ae′–Ag′). Closer views of the boxed areas in (Aa) are shown in (Ab)–(Ad); arrows in (Aa)–(Ad) indicate CD206+ cells (brown). (Ae–Ag′) Arrows and arrowheads indicate CD206+CD68+ double-positive and CD206+CD68− single-positive cells, respectively. (B) Double immunofluorescence staining of CD206 and CD163 corresponding to the area shown in (A). The arrow indicates a CD206−CD163+ single-positive cell; the inset is a closer view of the boxed area showing CD206+CD163− single-positive cells on a blood vessel. (C) Double immunofluorescence staining of CD206 and laminin α5-chains (LAMA5). Arrowheads indicate CD206+ cells. (D) Double immunofluorescence staining of CD163 and laminin α2-chains (LAMA2), and CD206 and CD163 on nerve fibers. Arrows indicate CD206+CD163+ double-positive macrophages; insets show single-channel images of cells indicated by an asterisk. (E) Immunohistochemical staining of CD206 (Ea and Eb), and double immunofluorescence staining of CD206 (Ec) and CD68 (Ec′) in an active carious tooth. (Eb) Closer view of the boxed area in (Ea). Strong (arrowheads) and weak (arrow) reactions of CD206 (brown) are shown. (Ec and Ec′) Arrows and arrowheads indicate CD206+CD68+ double-positive and CD206+CD68− single-positive cells, respectively. (F) Double immunofluorescence staining of CD206 and CD163 near the section shown in (E). (Fb and Fb′) Closer views of the boxed area in (Fa). Arrowheads indicate single-positive cells, and the arrow indicates a weakly double-positive cell. (G) Double immunofluorescence staining of CD163 and laminin α2-chains near the section shown in (F). (Gb) Closer view of the boxed area in (Ga). Arrows highlight the close association of CD163+ cells with laminin α2-chains (Ga and Gb). (H) Double immunofluorescence staining of CD206 and CD163 in a slowly progressing carious tooth. (Hb and Hb′) Closer views of the boxed area in (Ha). Arrow and arrowhead indicate CD206+CD163+ and CD206−CD163+ cells, respectively. (I) Graph showing the ratios of CD206+CD163+ double-positive/CD206−CD163+ single-positive macrophages in intact, active, or slowly progressing carious teeth. Data are presented as the mean ± SEM; n = 4 per group. ##p < 0.01, one-way ANOVA and Bonferroni tests. bv, blood vessel; nf, nerve fiber; ob, odontoblast; p, dental pulp; Sc, Schwann cell. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm except where indicated.

Distinct kinetic behavior of THP-1 macrophages cultured on laminin-211 or -511

The endothelial laminin-511 reportedly promotes monocyte differentiation to macrophages (15); based on the distribution patterns of laminin isoforms and M2-like macrophages in this study, we hypothesized that laminin isoforms may influence macrophage phenotypes. To test this hypothesis, we cultured THP-1 cell–derived macrophages, which are commonly used as an in vitro model of human macrophages, on plates coated with laminin-211 (a Schwann cell–associated laminin) or laminin-511 (a ubiquitous component present in the basement membranes of most tissues); plates coated with laminin-111 (a widely used laminin and a component of Matrigel) and non-coated plates were used as negative controls (Fig. 3A). PMA induced THP-1 monocytes to differentiate into macrophages (M0 macrophages). On the basis of morphology identification under phase-contrast microscopy, THP-1 cells plated on the distinct laminin isoforms showed different reactions to PMA incubation for 30 min. Most THP-1 cells plated on laminin-211 remained spherical with a 20-µm diameter, although some continued floating on the plate; cells plated on laminin-511 rapidly adhered and extended their cytoplasm onto the plate (Fig. 3B). At 1 and 2 d after PMA stimulation, THP-1 cells on laminin-211 appeared homogeneous with a spherical shape (Fig. 3B). In contrast, THP-1 macrophages cultured on laminin-511 were heterogeneous and comprised round, elongated, or expanded-shaped cells (Fig. 3B). In the absence of cytokines (control), the THP-1 macrophages grown on laminin-211 remained spherical (Fig. 3C). In contrast, macrophages cultured on laminin-511 had expanded cytoplasm (Fig. 3C). IL-4 stimulation promoted an elongated shape for cells cultured on laminin-211 and -511 (Fig. 3C). In contrast, IL-10 treatment altered the shape of macrophages grown on laminin-511 to a spherical appearance (Fig. 3C), whereas macrophages on laminin-211 remained spherical (Fig. 3C). Immunofluorescence staining indicated that CD206+ cells stimulated with IL-4 became elongated, but most CD163+ cells stimulated with IL-10 were spherical on laminin-211 and -511 (Fig. 3C). The THP-1 cells plated on laminin-111 or no-laminin coating showed flattened, elongated, or round appearances, both at 2 d after PMA stimulation (data not shown) and in control cultures (Fig. 3C). IL-4 stimulation promoted an elongated shape, whereas IL-10 stimulation altered the appearance to spherical, on both laminin-111–coated and non-coated dishes (Fig. 3C). qRT-PCR analysis of CD68 (a pan-macrophage marker) indicated no significant differences in control cultures (Fig. 3D). The level of CD206 mRNA expression was similar in control groups (Fig. 3D). IL-4 treatment significantly upregulated CD206 expression, and IL-10 treatment slightly increased CD206 expression; however, no significant differences were present in the dishes (Fig. 3D). In contrast, the mRNA expression of CD163 was induced in a distinct manner by individual laminin isoforms (Fig. 3D). CD163 expression was significantly higher when cells were grown on laminin-211 than when they were grown on laminin-111 or on non-coated dishes (control); IL-10 stimulation significantly increased CD163 expression, particularly on laminin-211 (Fig. 3D). In the presence of IL-4, the expression level of CD163 exhibited a minor change, compared with control cultures; the expression level of CD163 was significantly higher on laminin-211–coated dishes than on other dishes (Fig. 3D). Because differences in CD163 expression among cells grown on laminin-111, -211, and -511 were already detected in control cultures, we also investigated the level of IL-10 mRNA expression in control cultures. As expected, the level of IL-10 mRNA expression was significantly higher on laminin-211–coated dishes than on other dishes (Fig. 3D).

FIGURE 3.
FIGURE 3.

THP-1 macrophages cultured on laminin-211 or -511 demonstrate distinct kinetic behavior.

(A) Schematic illustration of the experimental method. (B) Morphology of THP-1 cells cultured on laminin (LN)-211/-511 in the presence of PMA. Arrowheads indicate flattened irregular, elongated, or expanded appearances on LN-511. (C) Morphology and immunofluorescence staining for CD206 and CD163 in THP-1 macrophages cultured on LN-211/-511/-111 and on no-laminin coating with IL-4 or IL-10. Asterisks show the cytoplasm of macrophages grown on LN-511. (D) Gene expression levels of CD68, CD206, CD163, and IL-10 in THP-1 macrophages grown on LN-211/-511/-111 and on no-laminin coating, with or without IL-4 or IL-10. Data are presented as the mean ± SEM; n = 3 sets of macrophage cultures. ns, not significant. #p < 0.05, ##p < 0.01, one-way ANOVA and Bonferroni tests. *p < 0.05, **p < 0.01, Student t test versus the corresponding control. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm.

Similar behavior of THP-1 macrophages cultured on laminin-211 or -511 to macrophages cultured on functionally analogous E8 fragments of laminins

The E8 domain of the laminin α-chain harbors principal integrin interaction sites (11). To validate the distinct kinetic behavior of THP-1 macrophages induced by laminin isoforms, we used recombinant minimum fragments containing the E8 domain. E8-α2 or E8-α5 were used to coat plates instead of laminin-211 or -511, respectively (Fig. 4A), and the experiments described above were repeated. The morphology alterations of THP-1 macrophages grown on E8-α2 and E8-α5 were similar to the alterations of THP-1 macrophages grown on laminin-211 and -511, respectively, in controls as well as in cells treated with IL-4 or IL-10 (Fig. 4B). qRT-PCR analysis showed similar results (Fig. 4C). No difference in CD68 expression was observed in controls when using E8-α2 or E8-α5. There were no significant differences in CD206 expression between cells cultured on E8-α2 or E8-α5, with or without IL-4. In contrast, macrophages on E8-α2 had significantly upregulated CD163 mRNA expression, compared with macrophages on E8-α5, with or without IL-10 treatment. Moreover, the level of IL-10 mRNA expression was significantly higher on E8-α2 than on E8-α5. Next, we performed flow cytometric analysis to evaluate the percentage of CD163+ cells in IL-10–treated cultures. THP-1 macrophages cultured on laminin-211/E8-α2 were weakly attached to the plate, whereas macrophages grown on laminin-511/E8-α5 adhered rigidly to the plate and were difficult to detach, despite the use of enzyme treatment. Thus, for flow cytometric analysis, we used thermosensitive cell culture dishes because they were reported to be beneficial for laminin E8 fragment coating (21). As expected, the percentage and mean fluorescence intensity of CD163+ cells were significantly higher in E8-α2 fragment cultures than in E8-α5 fragment cultures (Fig. 4D).

FIGURE 4.
FIGURE 4.

THP-1 macrophages cultured on laminin E8 fragments of α2 and α5.

(A) Illustration of the recombinant minimum fragments containing the E8 domain. (B) Morphology of THP-1 macrophages cultured on laminin E8 fragments of α2 (E8-α2) and α5 (E8-α5), with or without IL-4 or IL-10. Asterisks indicate the cytoplasm of macrophages grown on E8-α5. (C) Gene expression levels of CD68, CD206, CD163, and IL-10 in THP-1 macrophages on E8-α2 or E8-α5, with or without IL-4 or IL-10. Data are presented as the mean ± SEM; n = 3 sets of macrophage cultures. (D) FACS analysis of CD163+ cells cultured on thermosensitive cell culture dishes coated with E8-α2 or E8-α5, in the presence of IL-10. A representative gating plot shows the percentage of cells and the mean fluorescence intensity (MFI) of CD163 expression. Data are presented as the mean ± SEM; n = 3 sets of macrophage cultures. *p < 0.05, Student t test versus the corresponding control or between two groups. ns, not significant. Scale bars, 20 µm.

Alterations in cell shape and mRNA expression of CD206 and CD163 in THP-1 macrophages cultured on laminin-511 and treated with integrin α3– or integrin α6–neutralizing Abs

Laminins serve as ligands for integrins with isoform-specific affinities (11). Laminin-α2/E8-α2 exhibits high affinity for integrin α7β1 but demonstrates poor affinities for integrins α3β1 and α6β1; in contrast, laminin-α5/E8-α5 has the highest affinity for integrins α3β1 and α6β1 (11). Because integrin signaling can alter cell shape and affect gene regulation (22), we hypothesized that the high affinities of integrins α3β1 and α6β1 were involved in the expanded cytoplasm of THP-1 macrophages and the reduction of CD163 mRNA expression detected in cells grown on laminin-511. To test this hypothesis, we used functional inhibition of integrin α3 (ITGA3) and integrin α6 (ITGA6) (Fig. 5A). We initially analyzed the mRNA expression levels of laminin-related α3/α6/α7 integrins in THP-1 macrophages on non-coated, laminin-211–coated, or laminin-511–coated plates in the absence of neutralizing Abs (Fig. 5B). All laminin-related integrins were identified in THP-1 macrophages (Fig. 5B). The expression levels of ITGA3 and ITGA6 were significantly upregulated in cells grown on laminin-511, while the expression level of integrin α7 (ITGA7) was significantly upregulated in cells grown on laminin-211 (Fig. 5B). The mRNA expression of integrin β1, a pair of integrin α subunits, was also confirmed (data not shown). As expected, treatment with ITGA3- or ITGA6-neutralizing Abs did not alter the shape of THP-1 cells grown on laminin-211; those cells remained spherical (Fig. 5C). Furthermore, neither neutralizing Ab produced significant differences in the mRNA levels of CD260 and CD163 in cells grown on laminin-211, compared with isotype control Abs (Fig. 5D). In sharp contrast, ITGA3- or ITGA6-neutralizing Abs drastically altered the shape of THP-1 cells grown on laminin-511, as well as the mRNA levels of CD206 and CD163 in those cells. At 1 d of culture, spherical and elongated-shaped cells were mixed in the presence of ITGA3- or ITGA6-neutralizing Abs at 5 (Fig. 5E) or 10 µg/ml (data not shown), among cells grown on laminin-511. After 2 d, growth on laminin-511 with ITGA3-neutralizing Abs at 10 µg/ml produced elongated cells with much thinner cytoplasm; after 5 d, most of those cells had a spherical shape (Fig. 5E). Conversely, THP-1 macrophages treated with ITGA3-neutralizing Abs at 5 µg/ml exhibited a heterogeneous cell shape, including elongated cells with thinner cytoplasm (Fig. 5E). In contrast to ITGA3-neutralizing Abs, exposure to ITGA6-neutralizing Abs did not result in elongated cells with thinner cytoplasm (Fig. 5E). Furthermore, exposure to ITGA3-neutralizing Abs at 10 µg/ml led to significant upregulation of the mRNA expression levels of CD206, CD163, and IL-10, compared with ITGA3-neutralizing Abs at 5 µg/ml or the corresponding isotype controls (Fig. 5F). Furthermore, exposure to ITGA6-neutralizing Abs at both 5 and 10 µg/ml led to significant upregulation of the mRNA expression levels of CD206, CD163, and IL-10, compared with isotype controls (Fig. 5F). Exposure to ITGA6-neutralizing Abs at 10 µg/ml led to significant upregulation of the mRNA expression levels of CD163 and IL-10, compared with ITGA6-neutralizing Abs at 5 µg/ml; CD206 expression was higher upon exposure to ITGA6-neutralizing Abs at 5 µg/ml, compared with ITGA6-neutralizing Abs at 10 µg/ml (Fig. 5F).

FIGURE 5.
FIGURE 5.

The effects of ITGA3- or ITGA6-neutralizing Abs on THP-1 macrophages cultured on laminin-211 or -511.

(A) Experimental protocol. (B) mRNA expression levels of ITGA3, ITGA6, and ITGA7 in THP-1 macrophages cultured on laminin (LN)-211/-511 and on no-laminin coating without integrin-neutralizing Abs. Data are presented as the mean ± SEM; n = 3 sets of macrophage cultures. #p < 0.05, one-way ANOVA and Bonferroni tests. (C) Morphology of THP-1 macrophages cultured on LN-211 in the presence of ITGA3- or ITGA6-neutralizing Abs at 5 µg/ml. (D) mRNA expression levels of CD206 and CD163 in THP-1 macrophages cultured on LN-211 with ITGA3- or ITGA6-neutralizing Abs at 5 µg/ml or isotype control IgG. Data are presented as the mean ± SEM; n = 3 sets of macrophage cultures. There were no significant differences between the neutralizing Ab and the isotype control (Student t test versus the corresponding control). (E) Morphology of THP-1 macrophages cultured on LN-511 in the presence of ITGA3- or ITGA6-neutralizing Abs at 5 or 10 µg/ml. Arrows indicate elongated cells with thinner cytoplasm after culture on LN-511 in the presence of ITGA3-neutralizing Abs. (F) mRNA expression levels of CD206, CD163, and IL-10 in THP-1 macrophages cultured on LN-511 in the presence of ITGA3- or ITGA6-neutralizing Abs at 5 or 10 µg/ml, or isotype control IgG. Data are presented as the mean ± SEM; n = 3 sets of macrophage cultures. ##p < 0.01, one-way ANOVA and Bonferroni tests; *p < 0.05, **p < 0.01, Student t test versus the corresponding control. Scale bars, 10 µm.

Discussion

Lymphatic circulation is the key mode for transport of Ags and immune cells from peripheral tissues to lymph nodes (23). Interactions between lymphatic endothelial cells and leukocytes control the migration of immune cells to peripheral tissues (24). Although laminin isoforms modulate cellular phenotypes in a tissue-specific manner (14), laminin isoforms in lymphatic vessels are poorly characterized (25). We were surprised by the unexpected gene expression and protein localization of laminin α3 (Fig. 1C, E) because it is typically expressed by epithelial tissues (12), including the developing dental epithelium (26). The vessels stained with laminin α3 were positive for CD31, an endothelial marker; however, they were not accompanied by αSMA-expressing pericytes or vascular smooth muscle cells (16), indicating that the vessels were not blood vessels. Because the laminin α3 staining was colocalized with LYVE1, a marker of lymphatic vessel endothelium, the vessels stained with laminin α3 were considered to be lymphatic vessels. Lymphatic vessels are absent in some tissues (27), and the existence of lymphatic vessels in dental pulp has been debated for many years (28–30), probably because of the lack of reliable lymphatic endothelial markers. Although the lymphatic hyaluronan receptor LYVE1 is a typical marker of lymphatic vessel endothelium, LYVE1 is also expressed in certain subsets of M2-like tissue macrophages (31, 32), including pulp tissue macrophages (29). Therefore, the LYVE1+ laminin α3− cells detected in the current study were considered to be macrophages. The laminin γ2-chain, together with α3- and β3-chains, constitutes the previously named laminin-5 (laminin-332); therefore, laminin-332 is presumably the laminin isoform of the pulpal lymphatic vessels. Recently, β4 integrin, the main receptor for laminin-332, was identified on macrophages (33). The interaction between laminin-332 expressed by lymphatic endothelial cells in breast cancer and integrin β4+ macrophages was reported to be involved in lymphatic remodeling (33). Therefore, future studies should explore the potential functions of laminin-332 related to pulpal lymphatic tissue integrity.

The current findings indicated phenotypically different M2-like macrophage subsets; CD206+CD163+ double-positive macrophages were predominantly detected in healthy dental pulp, whereas CD206−CD163+ single-positive macrophages were significantly increased in active carious lesions. The two subsets of macrophages have been reported in other animal tissues (34). CD206+CD163+ double-positive macrophages are long-lived and predominant in healthy uninfected tissues; in contrast, CD206−CD163+ single-positive macrophages are presumed to migrate from the blood and persist for short periods of time (34). Nerve-associated tissue macrophages are generally long-lived and maintained via endogenous proliferation; thus, they are markedly distinct from monocyte-derived macrophages that invade after injuries (9). Overall, the present data suggest that the CD206+CD163+ double-positive macrophages are long-lived tissue macrophages in human dental pulp, which provide essential cures for nerve homeostasis. Furthermore, CD206−CD163+ single-positive macrophages detected under acute carious lesions might be transient and derived from the blood.

Macrophage polarization is thought to be regulated by soluble factors; however, the physical properties of the adhesive interactions with the extracellular matrix, integrins, and cytoskeletal organization also modulate macrophage phenotypes (35). In human dental pulp, CD163+ macrophages are colocalized with Schwann cells in healthy and carious teeth (10), and the current study indicated that Schwann cells express laminin α2. Laminins influence cell adhesion, differentiation, and phenotype maintenance; thus, laminin-based cell cultures have been used to analyze their effects on cell behavior (14). In this regard, we analyzed the effects of laminin isoforms on macrophage differentiation and polarization in vitro. In contrast to CD206, the expression of CD163 mRNA and its protein were differentially regulated by the laminin isoforms. Notably, differences in the expression patterns of CD163 and IL-10 were already detected in control cultures; both were highest on laminin-211/E8-α2 (Figs. 3D, 4C). This implies that laminin α2 promotes the transcription of CD163 mRNA in an IL-10–dependent manner. These in vitro data suggest that Schwann cell–associated laminin is an inducible matrix of CD163+ macrophages.

Laminin-related integrins α3/α6/α7 were all expressed in THP-1 macrophages grown on laminin-211/511–coated plates and on no-laminin coating. The higher expression levels of ITGA3 and ITGA6 in macrophages grown on laminin-511, and ITGA7 in macrophages grown on laminin-211, were presumed to reflect their distinct affinities for laminin α-chains (11). Laminin α5 has particularly high affinities for α3β1 and α6β1 integrins, in contrast to the lowest affinity of laminin α2 (36, 37). In the current study, THP-1 cells plated on laminin-511/E8-α5 began to adhere to the plate within 5 min. Conversely, most THP-1 cells plated on laminin-211/E8-α2 continued floating with a spherical appearance 30 min later; the cell shape remained spherical thereafter. In addition, exposure to ITGA3- and ITGA6-neutralizing Abs did not affect the cell shape or mRNA expression levels of CD206 and CD163, compared with isotype control Abs. These findings were presumably because laminin-211 lacked affinities for α3β1 and α6β1 integrins. In sharp contrast, macrophages grown on laminin-511 exhibited substantial changes in both cell shape and mRNA expression in the presence of the neutralizing Abs. Exposure to both ITGA3- and ITGA6-neutralizing Abs led to the upregulation of CD163 mRNA expression, which could have been caused by the increase in IL-10 expression. Furthermore, exposure to ITGA3-neutralizing Abs led to a more rounded cell shape after 5 d, similar to the change that occurred when IL-10 was added to cells grown on laminin-511 (Fig. 3C). These results suggest that integrins α3 and α6 inhibit the expression of CD163, consistent with the observation that laminin-211, which has poor affinities for integrins α3 and α6, caused CD163 upregulation relative to laminin-511; moreover, Schwann cells expressing laminin-211 are accompanied by CD163+ macrophages. Additionally, IL-10 upregulates both CD163 and CD206, which may be linked to the upregulation of CD206 in the presence of ITGA3- and ITGA6-neutralizing Abs. Notably, ITGA6-neutralizing Abs at 5 µg/ml significantly increased CD206 expression, compared with ITGA6-neutralizing Abs at 10 µg/ml; this contrasted with the IL-10 expression pattern. Considering that the expression of CD206 mRNA was not affected by laminin, regardless of exposure to IL-4 or IL-10 (Fig. 3D), other factors may have effects on CD206 expression.

Taken together, it is reasonable to suggest that the laminin α2 expressed by Schwann cells may be involved in the maintenance of the CD163+ macrophage phenotype because of its low affinities for integrins α3β1 and α6β1. Because laminin α2 is densely distributed in the periphery of the dental pulp, where bacteria first attack during tooth decay or trauma, this molecule may protect nerve fibers and affect dental pulp immunity by influencing macrophage phenotypes.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Edanz for editing a draft of this manuscript. We thank Prof. Kiyotoshi Sekiguchi (Division of Matrixome Research and Application, Institute for Protein Research, Osaka University) for thoughtful and creative discussions.

Footnotes

  • This work was supported by Japan Society for the Promotion of Science KAKENHI Grants 16K11546 and 19K10146 (to N.Y.) and 16H05516 (to K.Y.).

  • Abbreviations used in this article

    E8-α2
    laminin E8 fragments of α2
    E8-α5
    laminin E8 fragments of α5
    ITGA3
    integrin α3
    ITGA6
    integrin α6
    ITGA7
    integrin α7
    LYVE1
    lymphatic vessel endothelial hyaluronic acid receptor 1
    qRT-PCR
    quantitative real-time PCR
    αSMA
    α-smooth muscle actin

  • Received December 3, 2021.
  • Accepted December 3, 2021.

This article is distributed under the terms of the CC BY 4.0 Unported license.

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