Integrin alpha-v/beta3 expression in equine lungs and jejunum

Abstract

Integrin alpha-v/beta3 (αvβ3) recognizes arginine-glycine-aspartic acid (RGD) sequences and has important functions in cell adhesion, signaling, and survival. However, the expression of integrin αvβ3 in the equine lungs and jejunum is not well understood. The objective of this study was to explore the hitherto unknown expression of integrin αvβ3 in the lungs and jejuna of the horse using light and electron immunocytochemistry. Immunohistochemistry showed integrin αvβ3 on the epithelium, the immune cells in Peyer’s patches, the smooth muscle, and the endothelium of equine jejuna. In equine lungs, we recognized integrin αvβ3 on the endothelium of blood vessels, the alveolar septa, the bronchial lymph nodes, and the cartilages, although the expression of integrin αvβ3 was weak on the epithelium of bronchioles. In conclusion, these are the first data to show the expression of integrin αvβ3 in equine lungs and jejuna.

Introduction

Integrins are heterodimeric transmembrane receptors comprised of 2 subunits, alpha (α) and beta (β). Integrins are present in many species, such as mammals, chicken, zebrafish, sponges, the nematode Caenorhabditis elegans (2 α and 1 β subunits, forming 2 integrins), and the fruit-fly Drosophila melanogaster (5 α and 1 β subunits, forming 5 integrins) (1). The integrin family has 18 α and 8 β subunits, forming 24 heterodimeric transmembrane receptors (1,2). All 5 integrins αv, 2 integrins β1 (α5β1, α8β1), and αIIbβ3 can recognize arginine-glycine-aspartic acid (RGD) peptide ligands, known as a general integrin-binding motif (2). Each subunit has an extracellular domain, a single transmembrane region, and a short cytoplasmic domain (3).

The extracellular domain, a ligand-binding site, transmits signals from outside into the cell interior and then receives the intracellular signals in reverse to regulate back to the affinity of their ligand-binding site (1,2,4,5). Although smaller than the extracellular domains, the cytoplasmic domains up-regulate the activation of integrins by their association with adaptor proteins, including Src, focal adhesion kinase, integrin-linked kinase, kindlin, paxillin, talin, and vinculin (6,7). Consequently, these interactions rearrange the cytoskeleton, thus affecting the structure and function of extracellular domains (6). The activation state of integrins is characterized by separation, twisting, pistoning, and hinging of their tails (6). The integrins with a highly bent physiologic conformation have low affinity for binding biological ligands (8).

Integrin alpha-v/beta3 (αvβ3), known as a vitronectin receptor, plays an essential role in cell adhesion, cell signaling, cell survival, angiogenesis, and leukocyte migration (9,10). The expression of integrin αvβ3 is increased in neovascular endothelial cells (10). This protein serves as a marker on the cell surface, which recognizes and binds peptides containing RGD (9,10). Our lab reported that integrin αvβ3 is expressed on the bronchial vasculature in the lungs of calves and dogs, as well as in the small intestine of calves, dogs, and pigs (11).

Horses suffer from many inflammatory diseases, including some of infectious origin (12,13). The mechanisms of inflammatory cell recruitment, cell activation, and changes in vascular permeability underlying diseases such as colic and viruses, as well as bacterial enteritis and pneumonia, are not fully understood (12,14–16). Diarrhea commonly occurs in horses and the altered gut barrier function results in increased secretion of water into the intestine (16). Integrin αvβ3, which plays a role in fundamental processes, such as cell signaling, cell migration, and vascular hydraulic conductivity (17,18), may function in equine inflammatory diseases of the lung and the intestine. No data presently exist on the expression of integrin αvβ3 in equine tissues. The objective of this study was therefore to explore the expression of integrin αvβ3 in the lungs and jejuna of horses.

Materials and methods

Materials

Jejuna and lungs from horses (n = 4 each) were processed and embedded in paraffin blocks at Western College of Veterinary Medicine at the University of Saskatchewan. Mouse monoclonal integrin αvβ3 antibody (Clone LM609; Chemicon International, Temecula, California, USA), polyclonal goat anti-mouse immunoglobulins/horseradish peroxidase (HRP) secondary antibody (Dako, Santa Clara, California, USA), primary antibody polyclonal rabbit antihuman von Willebrant Factor (vWF) (Dako, Burlington, Ontario), Vector VIP peroxidase substrate kit for peroxidase (Vector Laboratories, Burlingame, California, USA), and methyl green (Dako) were purchased commercially.

Immunohistochemical staining of integrin αvβ3

Equine lungs and jejuna in paraffin blocks were sectioned at 5 μm thickness for immunohistochemistry as per a previous protocol (11). Briefly, sections were deparaffinized, followed by quenching the endogenous peroxidase activity and treatment with pepsin. After 2 h of blocking with 1% bovine serum albumin (BSA), the sections were incubated with monoclonal integrin αvβ3 antibody (Clone LM609; Chemicon International) and appropriate polyclonal goat anti-mouse immunoglobulins/HRP secondary antibody. The color development was carried out with Vector VIP peroxidase substrate kit. The controls included staining with the anti-endothelial marker vWF serving as a positive control or isotype antibody matching control [mouse immunoglobulin (IgG1)] instead of primary antibody or the omission of primary antibody serving as negative controls.

Immunogold electron microscopy for integrin αvβ3

Equine lungs and jejuna in LR white resin were sectioned at 100 nm thickness on nickel grids. The nonspecific bindings were blocked by BSA 1% in Tris-buffered saline before 1-hour incubation in the integrin αvβ3 antibody (50 μg/mL). A section stained without primary antibody was used as a negative control. After being washed 3 times in Tris-buffered saline, the sections were incubated for 1 h in 15–nm gold-conjugated anti-mouse secondary antibody with 1:100 dilution. Samples were then incubated in 2% aqueous uranyl acetate, an indicator for negative staining, then Reynold’s lead citrate to enhance the electron-scattering properties of biological components inside the cells. The micrographs were imaged using a transmission electron microscope (Hitachi HT7700, XFlash 6T160; Munich, Germany) operated at 80 KV.

Results

Integrin αvβ3 was detected on the equine lungs and jejuna. Immunohistochemistry showed integrin αvβ3 on the endothelium of blood vessels, the alveolar septa, the bronchial lymph nodes, the cartilages, the epithelium of bronchioles in equine lungs, neutrophils, type-1 epithelium, and pulmonary intravascular macrophages (Figures 1, 2). Immunoelectron microscopy yielded data on the detailed subcellular expression of the integrin. We found that integrin was present in type-I alveolar epithelial cells, pulmonary capillary endothelium, and neutrophils (Figure 2). The integrin was localized on the plasma membrane, cytoplasm, and the nuclei of these cells. Pulmonary intravascular macrophages also expressed integrin αvβ3 on their surface, cytoplasm, and the nucleus (Figures 2B, C).

Figure 1.

Equine lung immunohistochemistry for integrin αvβ3. Lung sections from healthy normal horses, stained with BSA (B) or IgG1 isotype control (D), showed only the green-blue color of methyl green counterstaining, while vWF antibody (F) reacts with vascular endothelium (purple-pink color). Integrin αvβ3 staining (arrows to purple-pink patchy deposition) is observed in alveolar (Av) septum (C), bronchiole (Br) epithelium (A), endothelium on blood vessel (BV, C), bronchial lymph node (LN, E), and cartilage (Ca, E). Magnification: 400×.

Figure 2.

Immunogold electron microscopy for integrin αvβ3 of an equine lung. The transmission electron micrograph of the normal healthy equine lung showed staining for integrin αvβ3 (arrows) in the plasma membrane, nucleus (N), and cytoplasm of a neutrophil in a capillary (A), a pulmonary intravascular macrophage (PIM) (B and C show the boxed area highlighted), a type-1 epithelium on alveolar septa (EP in A and B), and endothelium (E in A and B). The PIM was characterized as a giant and irregularly shaped leukocyte with a kidney bean-shaped nucleus adhering to the capillary endothelial cell on the thicker side of the alveolar septum. The cytoplasm of PIM contains some vacuoles. As — alveolar space; Bv — blood vessel; E — endothelium; EP — epithelium; N — nucleus. Magnification: A — 15 000× and B — 10 000×.

In equine jejunum, integrin αvβ3 was found in the epithelium, lymphocytes in Peyer’s patches, smooth muscles, and endothelium lining of the lumen (Figure 3). Negative controls for the specificity of primary or secondary antibody included mouse IgG1 isotype control instead of integrin αvβ3 antibody or the omission of primary antibody or both primary antibody and secondary antibody conjugated HRP. All negative controls showed only the green-blue color of methyl green counter staining, but no positive staining reaction (Figure 4).

Figure 3.

Equine jejunum immunohistochemistry and immunogold for integrin αvβ3. Immunohistochemistry results show positive staining integrin αvβ3 (purple-pink) on epithelium on lumen (A), glands (C), endothelium (D), cells in Peyer’s patches (E), and smooth muscle (F). The immunogold electron micrograph of integrin αvβ3 expression on the epithelium of normal healthy horse jejunum shows integrin αvβ3 on the nucleus, cytoplasm, and apical surface of the epithelium (B). L — lumen; EP — epithelium; BV — blood vessel; SM — smooth muscle; G — gland; P — Peyer’s patches; N — nucleus. Magnification: A, C–F — 400× and B — 20 000×.

Figure 4.

Integrin αvβ3 immunohistochemical staining controls of equine jejunum. From top to bottom, the equine jejunal section, stained with BSA and omitted integrin αvβ3 primary antibody, showed only the green-blue color of methyl green counterstaining. The equine jejunal section, stained with IgG1 rabbit isotype control instead of integrin αvβ3 primary antibody, also served as negative control and did not show any positive reaction. The equine jejunal section, stained with vWF antibody, served as a positive control for the protocol and reacted only with the vascular endothelium (pink-violet). Magnification: 400×.

Discussion

Financial losses due to intestinal and respiratory diseases in horses range in the billions of dollars every year worldwide (16). The lung and intestine are 2 organs of the body that constantly interact with the environment through air and food, respectively, and are exposed to pathogens and allergens (14,15). The unique and heterogenous lining of the epithelium in the intestine and the lungs is the first physical, physiological, and immunological barrier, with a well-developed system for handling bacteria and viruses (14,15). The epithelia employ various molecular strategies to tackle invading microbes and interaction with the intestinal microbiota.

We have previously provided evidence on the expression of integrin αvβ3, which is an important adhesion and signaling molecule in rats, calves, pigs, dogs, and humans (11,17,19,20). In the present study, we continued exploring the specific and detailed expression of integrin αvβ3 on the lungs and jejunum of horses, where we found that integrin αvβ3 was present on the epithelium, endothelium, and immune cells.

We have previously reported on the occurrence of integrin αvβ3 on porcine intestinal epithelia, as well as on the airway and alveolar epithelia of the lungs of pigs, dogs, and cattle (11). Integrin αvβ3 may play an important role in the adhesion and uptake of bacteria and viruses by the epithelial cells and aid in keeping the epithelium clean (21). Recently, there has been major interest in understanding the formation and ecology of the microbiome in various organs, including in the gut and airways of animals (12,14–16). The role of adhesive proteins as integrin αvβ3 in maintaining healthy microbiome and the interactions of microbiome with the equine epithelia remain unexplored and may be an area of interest. This could be especially important in species such as horses that have a high incidence of intestinal diseases, such as colic (12) and virus infection (16), as well as lung diseases such as heaves (13). Similar to these data, we also previously demonstrated the expression of integrin αvβ3 on the bronchiolar epithelia and alveolar macrophages in normal human lungs (20). The expression of integrin αvβ3 was intense in alveolar macrophages in human sepsis lungs (20).

There are differences in the anatomy and physiology of the lungs of horses and other mammals including humans since horses’ lungs support intense athletic performance (22). Horses’ lungs are also populated with pulmonary intravascular macrophages (PIMs), which humans do not have, in addition to the usual complement of alveolar macrophages (23,24). These PIMs have an electron-dense globular surface-coat (23). It has previously been shown that PIMs in horses express TLR4 (25), which could explain why horses have an enhanced sensitivity to lipopolysaccharide endotoxin-induced lung injury (25–27).

In this study, we show the presence of integrin αvβ3 in PIMs. Considering the role of integrin in cell signaling, it is possible that the integrin αvβ3 provides another mechanism for activating PIMs. It is important to note that depletion of PIMs, even in a spontaneous disease such as heaves, reduces lung inflammation and clinical signs of the disease (28,29). Pulmonary intravascular macrophages (PIMs) play an essential role in removing circulating particles in the capillaries (30) and the immune system in the lungs (31,32). There are recent data showing that when PIMs are induced in rodents, which normally lack PIMs, the induced PIMs increase susceptibility to lung inflammation and that this susceptibility ends when the PIMs are depleted (33). More studies are needed to investigate the occurrence of PIMs in humans (32). In this study, the expression of integrin αvβ3 in equine PIMs provides a potential mechanism of activation for PIMs. Integrin αvβ3 may play a role in the aggregation of platelets around PIMs in equine lungs as has been reported previously in the lungs of cattle infected with Mannheimia hemolytica (29).

In this study, we also found integrin αvβ3 on neutrophils. Some earlier data have shown that integrin αvβ3 is important in the locomotion of neutrophils (34–37). Moreover, in this study, the occurrence of integrin αvβ3 on lung vascular endothelium is consistent with previous findings and may play a role in maintaining endothelial barrier function as demonstrated in previous studies (18,38,39), as well as in angiogenesis (40).

Taken together, the findings of this study are the first elucidation of the cellular and subcellular expression of integrin αvβ3 in equine lungs and jejunum. Considering the role of integrin αvβ3 in cell biology and signaling, this sets the stage for further studies to assist in better understanding the expression and biology of integrin in the inflamed lungs and intestines of horses.