Neutrophil recruitment to the gastrointestinal tract

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CURRENT Neutrophil

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ROBERT A. KOZOL, M.D.l Wayne

State

University

School of Medicine, Detroit, Southfield & Outer Drive, Submitted

Michigan, and Veterans Administration Allen Park, Michigan 48101

for publication

The infiltration of an organ or tissue by neutrophils is the hallmark of acute inflammation. Recent work from many laboratories suggests that neutrophils may play a role in the development of tissue injury in a variety of disease states in the gastrointestinal tract. These diseases include gastritis, necrotizing enterocolitis, ileitis, ulcerative colitis, and ischemia reperfusion injuries. In view of this recent interest in the neutrophil and its relationship to GI diseases, it seems timely to review what is known about neutrophil recruitment to the gastrointestinal tract. This review will therefore focus on the sojourn of the neutrophil from the circulation to its destination in the GI tract. o 1992 Academic PRESS, IIW.

Neutrophils originate in the bone marrow and are delivered to the peripheral circulation with a half-life of 7 hr in the blood stream or 2 to 3 days in tissue. Neutrophils may be destroyed in the reticuloendothelial system or in situ by apoptosis and ingestion by macrophages [13]. The polymorphorphonuclear leukocyte (PMN)2 may be influenced by a litany of chemoattractants (Table 1). A chemoattractant is a molecule which stimulates PMN locomotion. Neutrophil chemotaxis is the directed movement of the neutrophil toward a chemoattractant. As elucidated primarily in studies utilizing the neutrophilchemotacticfactorformyl-methionyl-leucyl-phenalalanine (f-MLP) and its receptor [14], neutrophil chemotaxis is triggered by the binding of a chemoattractant (ligand) to its receptor on the neutrophil plasma membrane. This ligand-receptor complex then interacts with a transmembrane signal protein or so-called G-protein [XI]. The activation of the G-protein results in a cascade of biochemical events leading to the remodeling of the i Dr. Kozol is a funded investigator of the Veterans Administration. ’ Abbreviations used: PMN, polymorphonucleocyte; f-MLP, formyl-methionyl-leucyl-phenylalanine; NCA, neutrophil chemotactic activity; NCF, neutrophil chemotactic factor; PAF, platelet activating factor; LTB,, leukotriene B-4; IBD, inflammatory bowel disease; NEC, necrotizing enterocolitis; TNB, trinitrobenzene sulfonic acid. 0022-4804/92 Copyright All rights

$4.00 0 1992 by Academic Press, of reproduction in any form

310 Inc. reserved.

April

Medical

Center,

2, 1991

plasma membrane, changes in cytoskeletal organization, and in polarization of the neutrophil. Specifically, the neutrophil transforms from spherical to ovoid with a “head” and a “tail” [16]. The cell is thus prepared to migrate. Migration begins with PMN adherence to endothelial cells in the microvasculature. In fact, neutrophil delivery to the GI tract may be blocked with monoclonal antibodies to adherence-promoting leukocyte glycoprotein, CD18 [17]. PMN movement is dependent on the expression of receptors for chemoattractants, chemoattractant concentration, the presence of chemotactic inhibitors, and by the terrain (“contact guidance”). Contact guidance is defined as the influence of the alignment of the tissue substratum on the locomotion of cells [181. An example of regulation by receptor expression is seen with the bacterial-derived chemoattractant f-MLP [19]. In low concentrations (at a distance from the tissue injury) f-MLP binds high affinity receptors inducing PMN locomotion. As the cell approaches the inflammatory site, f-MLP is present in higher concentrations and binds low affinity receptors inducing cytotoxic functions. In addition, the presence of one chemoattractant may “up regulate” or “down regulate” receptors for a different chemoattractant [201. Neutrophil recruitment may be instigated by several mechanisms (Fig. 1). One mechanism is nonspecific, with chemotaxis stimulated by products of tissue and cell injury such as that seen with a soft tissue crush injury. Examples of other mechanisms include neutrophil recruitment due to factors produced by bacteria (such as f-MLP) and recruitment toward immune cell-derived factors such as leukotrienes. Neutrophil migration has been studied by a variety of in viva and in vitro techniques. In the laboratory, the most common method has been the multiwell chamber, micropore filter technique (modified Boyden chamber). In this technique, test solutions or control solutions are placed in the bottom compartment and the neutrophil suspension is placed in the top compartment of each well. The compartments are separated by a filter with a 3- to 8-pm pore size. The plates are incubated at 37°C for

ROBERT

TABLE Partial

List

A. KOZOL:

NEUTROPHIL

1

of Putative Neutrophil Factors

Chemotactic

Reference(s)

Chemoattractant C5a 11-S LTB-4 f-MLP High molecular wt. factors Macrophage inflammatory PAF

161 [71

181 191 WI protein-2

1111 1121

an hour to allow neutrophil migration through the filter. The filters are then stained and counted under the microscope [21]. Data are presented either as numbers of neutrophils at various distances in the filter (chemotactic index) or as the number of neutrophils that have entirely transgressed the filter. Another technique involves neutrophil labeling with radioisotopes followed by counting for radioactivity at the target site [22]. Radio-labeled neutrophils and counting can be used both with filters (in uitro) and in vivo and is the principal behind the “tagged white blood cell scan.” Less commonly used techniques include migration on agar [23] or direct visualization under cover slips [24]. With this overview in mind, we will now examine information concerning neutrophil recruitment according to GI tract segment. Specific areas covered will be stomach, small bowel, and colon. STOMACH

With its location in the proximal gastrointestinal tract, the stomach is exposed to undiluted chemicals, pharmaceuticals, and pathogens that are intentionally or accidentally ingested. With this variety of insults it would not be surprising to find that neutrophils are recruited to the gastric mucosa by several mechanisms. Of the various gastritides, that associated with the bacterium Helicobacter pylori is characterized by the greatest infiltration of PMNs into the gastric mucosa [25]. Supernatants from H. pylori cultures contain neutrophil chemoattractants with a molecular weight of about 10,500 Da [26, 271. It is interesting to contrast human type B gastritis caused by H. pylori with gastritis in ferrets caused by Helicobacter mustelae. The H. mustelae gastritis is not characterized by a marked PMN response. In culture H. mustelae lacks the 10,500 Da chemotactic substance seen with H. pylori [26]. These differences support the probability that the NCF seen in culture from H. pylori is probably responsible for PMN recruitment in H. pylori-induced gastritis. Platelet activating factor (PAF) is a known neutrophil chemoattractant which causes gastric mucosal injury in

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laboratory animals [281. Mucosal injury induced by PAF is neutrophil dependent [29]. The PAF antagonist CV3988 protects against gastric injury in an ischemia-reperfusion model in rats [30]. Conversely, the PAF antagonist WEB 2086 offered no protection against nonsteroida1 anti-inflammatory-induced ulcers in rats, despite the fact that this injury is also neutrophil dependent [31]. PAF remains a candidate chemoattractant in some cases of gastric mucosal injury. Leukotrienes have been implicated in the pathogenesis of ethanol-induced hemorrhagic lesions in the gastric mucosa [32]. Evidence against this role is the fact that inhibition of leukotriene synthesis failed to protect against ethanol-induced hemorrhagic lesions in a rat model [33]. It is important to note that hemorrhagic erosions are early lesions and neutrophils may not be involved in their development. In contrast, neutrophils have been suggested to play a role in ethanol-induced gastric mucosal injury involving inflammation [34]. Ethanol can induce the production of leukotrienes by gastric mucosa [35]. Leukotriene B-4 is a particularly potent neutrophil chemoattractant and may be responsible for neutrophil recruitment in some casesof gastritis. Ethanol can also cause degranulation of gastric mucosal mast cells [36] which are known sources of LTB, [37] and other chemoattractants [381.These studies offer several potential pathways for neutrophil recruitment after mucosal injury due to ethanol. In addition, rabbit gastric tissue appears to release a chemotactic inhibitor from the serosal surface after mucosal exposure to ethanol [39]. Thus, it is possible that the stomach may be responsible for chemotactic inhibitors detected in the serum of alcoholic patients. Isolated gastric tissue is capable of releasing high molecular weight neutrophil chemoattractants (NCF) [40]. High molecular weight NCFs have also been isolated from human duodenum [41]. These high molecular weight NCFs are biochemically similar to those seen in the sera of patients with a variety of allergic diseases

ORGAN PARENCHYMAL CELL PROTEASES. PRODUCTS OF TISSUE INJURY 1 non - specific (tissue injury)

FIG. 1. cific

I fMLP 1 BACTERIA DERIVED

\ interleukins. leukotrienes

1 IMMUNE CELL DERIVED (includes PMN, MonoCyte 8 MAST cell)

Mechanisms for neutrophil recruitment and specific mechanisms (see text).

including

nonspe-

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ACTIVATING FACTOR

NER

HIGH MOLECULAR

WEIGHT NCF

FIG. 2. The mast cell as a source of multiple chemoattractants. The precise determinants (afferent signals) responsible for specific mediator release by the mast cell are currently unknown [68].

[lo]. It is of interest that basophils and mast cells are sources of high molecular weight NCFs. Takemura et al. have shown that compound 48/80 which degranulates mast cells induces gastric mucosal injury in rats [42]. In addition, disodium cromoglycate, a mast cell stabilizing agent, protects against PAF-induced gastric damage [29]. Since PAF-induced gastric injury is neutrophil dependent, these studies and those concerning LTB, discussed above suggest a role for the mast cell in orchestrating PMN traffic in gastrointestinal inflammation (Fig. 2). Neutrophil chemotactic activity is elevated in gastric secretions of patients with gastritis compared to control patients [43]. Kozol et al. compared NCF activity in gastric secretions to gastric tissue PMN counts in the same patients (a signal to response comparison). Although the correlation between NCF activity and PMN counts is not linear, clear trends were established between the magnitude of gastritis histologically and the NCF levels [44]. SMALL

BOWEL

PMN movement from the circulation to the intestinal lumen has been demonstrated in inflammatory bowel diseaseand in gastroenteritis utilizing Indium-111 WBC scans [45]. Despite this, little information is available regarding the controlling mechanisms of this PMN movement. Studies involving intubation of the jejunum in patients with active ileal Crohn’s disease (but grossly and histologically normal jejunum) offer suggestions of early PMN involvement or perhaps a “preclinical” state. Specifically, Ahrenstedt et al. have shown high levels of complement components in jejunal fluid of such patients [46]. Complement components may effect neutrophil recruitment directly (C5a) or indirectly by activating other cytokine producing cells such as mucosal mast cells. It should be noted that circulating C5 levels are probably not elevated in patients with Crohn’s disease [47]. Using similar small bowel intubation, Hallgren et al. detected elevated levels of myeloperoxidase (a neutrophi1 granule constituent) in jejunal fluid of Crohn’s dis-

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ease patients [48], indicating early or preclinical PMN recruitment. Using a different approach, many investigators have sought a neutrophil defect in inflammatory bowel disease (IBD). Rhodes and Jewel1 found neutrophil chemotaxis to be normal in patients with ulcerative colitis or Crohn’s disease [49]. Reviewing available data, Elson has concluded that a PMN defect in IBD is unlikely [50]. In fact, the hypothesis of a PMN defect in IBD patients dodges the question of why, for example, the PMNs flock to the terminal ileum in Crohn’s disease. Anton et al. have demonstrated increased receptors for f-MLP on PMNs of patients with Crohn’s disease [51]. They believe, however, that this finding represents differences in PMN conditioning (environmental effects) rather than an intrinsic PMN defect. Formyl-methionyl-leucyl-phenylalanine (f-MLP) has been shown to increase intestinal mucosal permeability in rats [52]. As a peptide produced by Escherichia coli and other enteric flora, it may therefore not only be responsible for PMN recruitment to the gut (as a direct chemoattractant) but also may be responsible for permeability changes resulting in changes in antigen presentation and processing in the gut wall. Clearly this is an avenue for much future investigation concerning the pathophysiology of inflammatory bowel diseases. More will be said about f-MLP in the following section on colon. Necrotizing enterocolitis (NEC) is an inflammatory disease which results in necrosis of the intestine in neonates. Hypoxia is believed to play a role in the etiology of this disease. Caplan et al. have demonstrated increased levels of PAF from hypoxic small intestine in rats [2]. Furthermore, they have produced pathologic changes similar to NEC by directly perfusing PAF into the mesenteric artery in rats [53]. The recruitment of neutrophils may be the critical effect of PAF in this disease state. In a feline model of intestinal ischemia, Zimmerman et al. have shown increased levels of leukotriene B, (LTB,) from intestinal mucosa [54]. Enhanced LTB, release and the subsequent PMN recruitment could be blocked using various lipoxygenase inhibitors. In addition, elevated LTB, levels have been implicated in helminth-induced intestinal inflammation [ 551. LTB, can be produced by a number of cells including neutrophils themselves and mast cells. The work of Perdue et al. has shown that mast cell activation and concomitant elevations in LTB, correlate with mucosal inflammation induced by nematodes in rats [56]. LTB, has also been implicated in the pathogenesis of inflammatory bowel disease and will be discussed further in the following section. COLON

The gastrointestinal inflammatory disease with the most evidence for a PMN role in pathogenesis is ulcera-

ROBERT

A. KOZOL:

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tive colitis. Ulcerative colitis is characterized histologically by sheets of PMNs in the mucosa and submucosa. Colonic disease which is grossly and histologically similar to ulcerative colitis can be produced in rabbits, rats, and mice by the intraluminal instillation of f-MLP [57, 581. Chadwick et al. have demonstrated production of chemotactic peptides such as f-MLP by many strains of intestinal bacteria [59]. With E. cd present at concentrations of lo6 to lo8 organisms/g/wet wt of stool it follows that f-MLP and other chemotactic peptides will be present in the human colonic lumen. As in most theories of pathogenesis of IBD, the missing link is the inciting factor. In other words, if f-MLP is present in all colons, then what is different in the colon of the patient with ulcerative colitis? Possibilities include differences in bacterial strains, concentration of NCF, mucosal integrity (permeability differences), etc. Rachmilewitz et al. examined inflammatory mediator release and timing of release in colitis in rats [60]. In this model, colitis is induced by topical trinitrobenzene sulfonic acid (TNB) in ethanol. These investigators found that interleukin-1 (11-l) was the most sensitive and reliable indicator of colonic mucosal inflammation and that 11-l levels correlated with myeloperoxidase (PMN) activity. It is of interest that 11-l is no longer believed to be a direct chemotactic factor for neutrophils [61]. The presence of 11-l receptors on neutrophils does infer a role in modulation of inflammation, however [62]. Mucosal LTB, was modestly elevated in this model, while platelet activating factor was not elevated at all. The role of leukotriene B, has been studied in colitis in man and laboratory animals. Fox et al. have demonstrated enhanced mucosal mast cell activity in colon from patients with ulcerative colitis [63]. Specifically, these mast cell preparations had elevated levels of LTB,, prostaglandin D,, and histamine. In a follow-up study from the same laboratory, the therapeutic agent 5-aminosalicylic acid was shown to inhibit mediator release by stimulated human mucosal mast cells [64]. Lobos et al. found that the majority of chemotactic activity from colonic mucosa of IBD patients was lipid extractable and coeluted with LTB, on HPLC [65]. In a rat model of acetic acid-induced colitis, Sharon and Stenson found LTB, levels 50 times greater than in normal rat colon [66]. In view of the possibility of LTB, being an important chemotactic mediator in IBD, Fretland et al. looked at blocking the LTB, effect. This group administered the LTB, receptor antagonist SC-41930 to guinea pigs prior to the establishment of acetic acid-induced colitis. They found a significant drop in myeloperoxidase activity and less histologic damage in the SC-41930 group [67]. These findings support a role for LTB, in the recruitment of PMNs and the pathogenesis of IBD. In constructing a unifying inflammatory mediator hypothesis for ulcerative colitis, the following possibility is offered. An intrinsic defect in colonic mucosal permeability (or an unidentified insult) allows submucosal access

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to chemotactic peptides (such as f-MLP). This recruits and activates neutrophils to the colon resulting in tissue damage. The acute injury may then be promulgated by the production of other mediators such as 11-l and LTB, by resident cells, possibly mast cells stimulated by the ongoing inflammation. SUMMARY

Many laboratories have focused on the role of the neutrophil in gastrointestinal inflammatory diseases. Individual neutrophil chemotactic factors are being scrutinized in various diseasesin hopes of establishing new therapeutic approaches to these disease states. Recent work discussed in this article suggests that the mucosal mast cell proves to play a prominent role in controlling movement of inflammatory cells. While progress has been made regarding chemoattractants and PMN recruitment, other areas influencing PMN traffic have been virtually unexplored. In the GI tract the greatest gaps in our knowledge lie in how various disease states may affect the “terrain” (the alignment of cells and connective tissue) thus altering PMN recruitment by affecting “contact guidance.” REFERENCES 1.

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