**3. Epithelial cell phagocytosis**

The primary function of epithelial cells is to form a barrier between the internal organs and the external environment. As such these tissues have evolved to be relatively efficient in anti-pathogenic mechanisms, including the secretion of antimicrobial peptides, functional mucociliary clearance, and phagocytosis [6, 7]. The integumentary skin layer is perhaps the most obvious epithelial cell layer; however, the epithelium also lines internal organs and mucosal surface tissues, such as the respiratory tract, digestive system, genitourinary organs, and neuronal tissues, among others [95]. The physiological organization and structure of the epithelium can vary, even within the same organ system, for example, the pseudostratified epithelium that lines the proximal airways progressively changes to a simple squamous epithelium that lines the alveolar airspace [96, 97]. Despite the multiple structural phenotypes, the primary function of any epithelium is to form a barrier, a protective layer of epithelial cells connected by tight junctions [98]. Tissue-resident myeloid cells, such as macrophages, are often labeled as the first line of defense when it comes

#### *Non-Myeloid Cell Phagocytosis DOI: http://dx.doi.org/10.5772/intechopen.110583*

to invading pathogens; however, it could be argued that epithelial cells provide that initial functional defense [6].

Efferocytosis appears to be a function of practically all tissues and cell types [7, 73]. Relative to professional phagocytes. The removal of damaged or dying cells can leave the barrier exposed and prone to further damage or infection. As such epithelial tissues have a remarkable capacity for repair to maintain barrier integrity and homeostasis [98]. It is well established that the primary mechanism to eliminate apoptotic epithelial cells is through extrusion into the external apical lumen [6, 99]; however, epithelial cells also engage in efferocytosis [7]. Efferocytosis is particularly important for subapical apoptotic bodies or if the epithelial lumen does not have a functional system for debris removal such as mucociliary clearance in the airways.

Apoptotic epithelia can express a wide array of "eat me" signals with the most common being phosphatidylserine [21, 29, 34]. Recognition of apoptotic cells by epithelia is somewhat less understood; however, uptake of apoptotic bodies and recognition by phosphatidylserine receptors on the epithelial cells are acknowledged to be an integral part of this process [34]. Some studies provide evidence that the process of efferocytosis observed in epithelia is distinct from that of professional phagocytes and even from other phagocytic processes [74]. Epithelial cells have a relatively strong expression of PRRs with innate capabilities of recognizing exogenous PAMPs [5, 6, 100, 101]. Activation of PRRs can induce strong inflammatory responses including cytokine release [102], however mucosal epithelial cells must maintain a bio-symbiotic relationship with natural bacterial flora and control the potential for excessive inflammatory stimulation [103]. To achieve this, many of the PRRs are either intracellular [103–106] to recognize pathogens in the process of infecting the epithelium or on the surface of a polarized epithelium restricted to the basolateral surface [101, 107] to detect pathogens that have breached the epithelial barrier. PRRs expressed on epithelial cells include the Toll-Like Receptors (TLRs), the C-type Lectin Receptors (CLRs), the NOD-like Receptors (NLRs), and the RIG-I-like receptors (RLRs) [6, 108, 109]. It has also been proposed that PRRs can also engage in zipper phagocytosis, as integral parts of internalizing pathogenic stimuli, in addition to internalization of the receptor itself to control excessive inflammatory responses [110–112]; however, it is unclear if this pathogen-induced internalization is consistent with zipper phagocytosis or even conserved in nonmyeloid cells, in principle it is a possibility. Often the overarching inflammatory response is studied in isolation from that of any possible phagocytosis response. However, it is important to recognize that there is significant overlap and control of one by the other. Indeed, it has been reported that signaling factors, such as Rac1, are necessary for phagocytosis and the subsequent control of anti-inflammatory cytokine release, key to inflammatory resolution [30]. Further insights into epithelial cell phagocytosis may well be found in the study of inflammatory cytokine biology.

A common place for epithelial phagocytosis study can be found in the retinal epithelium of the eye [113]. Separated by the blood-retina barrier [114, 115], the retinal epithelium is able to maintain a certain level of immune privilege from circulating leukocytes [116]. Whilst there is evidence for resident and infiltrating myeloid cells in these tissues [117], it is primarily the retinal epithelium that maintains homeostasis through phagocytic functions [113]. Aside from immune recognition, phagocytosis by the retinal epithelium is important for the biological process of photoreception [118]. The distal portions of photoreceptors in the eye, known as "Photoreceptor Outer Segments" (POS) are in direct contact with the retinal epithelium [119] and rich in membranous discs packaged with proteins known as opsins [120], which are

photosensitive. Exposure to light bleaches opsins to allow for signal transduction [121]. Extended exposure to these opsin-rich discs results in phototoxic damage and mature discs are shed from the distal tip to allow for the synthesis of new discs [113]. The retinal epithelium is perpetually "ensheathed" around the distal tips of photoreceptors [122], which upon shedding are phagocytosed into the retinal epithelium [119, 123]. The phagosome undergoes phagolysosomal maturation, including acidification and breakdown of the photoreceptor distal tips [113]. This entire process allows for the maintenance of long-term photoreceptors with short-lived distal tips by the retinal epithelial cells in an immune-privileged tissue. The retinal epithelium represents a prime example of a nonmyeloid cell performing specialized phagocytosis as a primary function in the homeostatic maintenance of its niche.

Internalization of pathogens by mucosal epithelium is well documented [5, 22–27]. Epithelial cells utilize both zipper and trigger mechanisms to internalize invading pathogens and engage in phagocytosis [6]. After internalization of the pathogen, the maturation of the phagosome in epithelia is akin to that of professional phagocytes [59], including markers of maturation, phagosome acidification, and lysosomal fusion [124]. The primary difference lies in the speed and efficiency when compared to professional phagocytes [125]. Despite this lack of efficiency, the contribution of phagocytosis of epithelial cells is still remarkably significant when considering cell numbers and so the impact of epithelial cell phagocytosis in pathogen clearance should not be ignored, having distinctive implications in both homeostasis and disease.

### **4. Endothelial cell phagocytosis**

Like epithelial cells, endothelial cells also form a physical barrier, specifically in the walls of fluid systems, such as the circulatory and lymphatic systems [126]. These barriers comprise squamous endothelial cells, which form a single cell layer lining the entire system [126]. Their primary functions are to maintain the barrier and act as a filtration system for fluid-containing cells or substances into, and out of, the circulatory system [127, 128]. Significant cross talk occurs between endothelial cells and professional phagocytes as the endothelium allows leukocytes to cross through the barrier into tissues during times of infection and stress [129]. The concept of endothelial cells acting as phagocytes is not new, with some reports dating back as early as the 1920s [130]. Such a process is important for the endothelium to maintain circulatory homeostasis with effective phagocytic clearance mechanisms [129]. Phagocytosis is clearly an important function for endothelial cells to possess and execute efficiently, failure to do so can lead to serious complications such as stroke [131, 132]. Due to its importance, phagocytic clearance by endothelial cells has been termed "Angiophagy" [131, 132].

In situations of physical damage to endothelial tissue, endothelial cells can often be the first to encounter potentially pathogenic insults, particularly pathogens that enter circulation. Like epithelial cells, endothelial cells strongly express PRRs, including TLRs, NLRs, and RIG receptors [133–137]. During times of inflammation, endothelial cell PRR expression is increased [138], an important process for innate recognition of potentially invasive pathogens. It is also imperative for endothelial cells to recognize endogenous material, such as aged red blood cells, to both prevent and clear micro emboli blockages [139]. Endothelial cells express Lectin-like oxLDL receptor 1 (LOX-1), a transmembrane protein that is capable of recognizing these aged red blood cells that express phosphatidylserine [36]. Endothelial cells can also clear other cellular material, such as apoptotic cell bodies of circulating leukocytes, including that of

#### *Non-Myeloid Cell Phagocytosis DOI: http://dx.doi.org/10.5772/intechopen.110583*

circulating professional phagocytes, such as neutrophils [140], and do so *via* recognition of lactadherin [141]. Endothelial cells capable of recognizing and engulfing circulating cellular material is not just a function of cellular turnover homeostasis, but this is important in reducing coagulative activity.

Angiophagy, as a phagocytic process, can be considered distinct from other mechanisms such as efferocytosis, as a specialized method of clearing vascular occlusions, which may or may not have "eat-me" recognition molecules. In several organ systems, angiophagy of large particulates, such as blood clots and fibrin, has been observed by endothelial cells in microvascular capillary structures, releasing the phagocytosed particles into the basolateral parenchyma [132, 142]. While the overall result remains consistent, angiophagy efficiency can vary between different organs [142]. The biomechanical processes of angiophagy are not well understood. Studies have demonstrated that projections of the endothelial cell wall known as "lamellipodia" extend into the occluded lumen after extensive cellular remodeling [142]. Engulfment of the occluding body occurs within a few hours, relatively quickly when compared to the entire angiophagy process, which can take several days. Post engulfment, the occluding body is trafficked to the underlying tissue where it can be further processed, often by myeloid cells [142]. A more comprehensive characterization, beyond engulfment in angiophagy, is lacking although mechanisms of phagocytosis are certainly present. Further reports have demonstrated that microparticles are internalized and retained intracellularly without any impact on barrier integrity [143].

A common endpoint of phagocytosis in some professional phagocytes is antigen presentation. After a functional inactivation of the pathogen, components of the pathogen are "presented" on the cellular surface of the phagocyte and used to activate specific lymphocytes, to initiate adaptive immune responses. This specialized function of antigen presentation is typically associated with dendritic cells but is also observed in other myeloid cells. Interestingly, antigen presentation has been observed in endothelial cells [144, 145], and even express MHCII, typically restricted to professional antigen-presenting cells, as a result of inflammatory stimulation [146]. As endothelial cells are not professional antigen-presenting cells and lack migrating capabilities important for effective antigen presentation, it is somewhat unclear as to why endothelial cells have developed antigen-presentation capabilities. It has been postulated to be important for T-cell-specific trafficking to sites of infection and stress [144]. Either way, strong phagocytosis machinery is required to process and present antigens on the cell surface.

Phagocytosis for endothelial cells is an important homeostatic process that allows luminal vasculature to remain clear of blockages and underlying tissues to remain clear of potentially pathogenic infection. The process of angiophagy to allow the extravasation of occlusions, and restoring luminal perfusion is arguably unique to endothelial cells as a process that even myeloid cells do not possess. Further work on the capabilities of endothelial cell phagocytosis could well lead to a better understanding and even treatment options for serious acute macro and microvascular disease.
