Differential expression of substance P receptors in patients with Crohn's disease and ulcerative colitis

GASTROENTEROLOGY 1995;109:850-860

Differential Expression of Substance P Receptors in Patients With Crohn's Disease and Ulcerative Colitis

CHRISTOPHER R. MANTYH,* STEVEN R. VIGNA,* R. RANDALL BOLLINGER,*

PATRICK W. MANTYH, § JOHN E. MAGGIO, II and THEODORE N. PAPPAS* *Department of Surgery and tDepartments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina; ~Molecular Neurobiology Laboratory, University of Minnesota, Minneapolis, Minnesota; and ~lDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts

Background & Aims: Although clinical and pathological differences exist between Crohn's disease (CD) and ulcerative colitis (UC), distinguishing features are often absent, making diagnosis and treatment problematic. This study evaluated the differences in the expression of substance P (SP) receptors in patients with CD or UC. Methods: Tissue samples from patients with inflammatory bowel disease or control patients were obtained at surgery, processed for 12SI-SP binding, and analyzed by quantitative autoradiography. Results: Pa- tients with CD showed a massive increase in SP receptors in lymphoid aggregates, small blood vessels, and enteric neurons of the small and large bowel relative to controls. Six of 16 CD specimens had no pathological evidence of CD yet continued to express high concentrations of SP receptors. Pathologically positive patients with UC showed high concentrations of SP receptors on colonic lymphoid aggregates and small blood vessels but not enteric neurons. No increased SP binding was evident in clinically and pathologically quiescent UC colons and normal UC Ueostomy samples. Con- clusions: The increased expression of SP receptors on the enteric neurons of patients with CD distinguishes CD from UC. The persistent increased SP binding in pathologically normal CD tissue may indicate a subclinical disease state. SP receptor expression may have important diagnostic, etiologic, and therapeutic use- fulness in inflammatory bowel disease.

'nflammatory bowel disease (IBD) encompasses two , disease states, Crohn's disease (CD) and ulcerative co-

litis (UC), that have been separated by clinical, endoscopic, radiological, and pathological differences. 1 Cur- rently, CD represents an inflammatory condition affecting any part of the intestine (but most commonly is found in the distal small bowel and proximal colon) with skip lesions, fissures, granulomas, and transmural inflammation as pathological features. UC by definition involves only the colon (although backwash ileitis may occur) and is usually uniform and continuous in mucosal

inflammation. 2 Although extensive diagnostic criteria exist to differentiate CD and UC, no single, uniformly reliable diagnostic feature exists. 3 In addition, a significant percentage of patients with IBD are incorrectly diagnosed or cannot be categorized into either CD or UC. It has been estimated that 5%-15% of patients with IBD have indeterminate colitis, a term used when macroscopic and microscopic examinations reveal both types of disease of the colon but are not diagnostic of either. 44 Further- more, misdiagnosis of colonic IBD may result in improper treatment because CD and UC have distinct medical and surgical therapies/

Because of the potential difficulty in assigning a diagnosis of CD or UC, several investigators have attempted to find a distinct marker for either disease state. These have included differential expression of cytokines and their receptors s-~2 and neutrophil autoantibodies.13 How- ever, the specificities and sensitivities of these methods are inconclusive, and the significance of neutrophil autoantibodies in the pathogenesis of IBD remains obscure. 14

The cause of the uncontrolled inflammation in IBD is also unclear. Numerous alterations in the immune system are evident in CD and UC. Several cytokines and other proinflammatory mediators have been shown to be ele- vated in affected regions of CD and UC bowel. 15-1s How- ever, to our knowledge, no study has yet determined if unaffected, pathologically normal regions of IBD bowel show any up-regulation of proinflammatory substances. This observation may be particularly important in CD, in which skip lesions between affected regions occur. Additionally, CD has a recurrence rate of between 50% and 90% after surgical resection of one region of affected bowel. ~9 Potentially, changes in the receptor density of a proinflammatory mediator in pathologically normal tissue may provide a rational explanation for the indolent nature of CD.

© 1995 by the American Gastroenterological Association 0016-5085/95/$3.00

September 1995 SUBSTANCE P RECEPTORS IN IBD 851

In this report, we have examined substance P (SP)

receptor distribution in surgically resected specimens taken from patients with CD and UC. SP is an 1 I - a m i n o

acid neuropeptide belonging to the tachykinin family of

peptides that acts via a G protein-coupled seven trans-

membrane domain receptor designated the neurokinin 1 receptor. 2°'-~ SP has multiple effects throughout the cen-

tral nervous system and the periphery, including potent nociceptive and proinflammatory properties. 22 SP acts

both directly and indirectly on immune cascades and on

the vasculature to cause plasma extravasation, edema, and

pain. 23 We have previously shown that resected bowel

from both patients with CD and UC shows a > 1 0 0 0 -

fold increase over controls in SP receptor levels in active

CD and UC tissue in lymphoid aggregates and small blood vessels. 24 It was suggested that SP may contribute

to the inflammatory response in IBD via the dramatic

increase in its receptor density. However, in this study, no direct comparison of the regional distribution of the

SP receptor was performed. Additionally, we did not

examine asymptomatic patients with IBD or pathologi-

cally normal regions of resected IBD bowel for SP recep-

tor up-regulation.

The differential expression of SP receptors in IBD may be important not only for diagnostic and pathophysiolog-

ical purposes but also therapeutically. Recently, several

synthetic nonpeptide SP receptor antagonists have been developed that have been shown to block the inflammatory and nociceptive effects of SP in vitro and in vivo. 25

These antagonists have been shown to be nontoxic and

have been suggested for use in several human inflammatory diseases, including CD and UC. 26 Knowledge of the

gut SP receptor status of patients with CD and UC during active and quiescent disease would be crucial in de-

termining the t iming and duration of dosing of these

potential agents.

In the present study, we have examined 32 patients

with IBD with both active and quiescent disease states and 10 control patients. Regional differences in the localization of the ectopically expressed SP receptors were

compared in pathologically positive tissue samples from patients with CD and UC. In addition, we examined SP

receptor expression in pathologically negative surgical

specimens from patients who had a previous diagnosis

of CD or UC.

Materials and Methods

Patients

Tissue samples were taken from patients undergoing operative removal or reanastomotic procedures for IBD at Duke University Medical Center and the Durham Veterans Affairs

Hospital. A list of the patients with CD, patients with UC, and control patients studied is provided in Table 1. A total of 16 patients with a previous diagnosis of CD and 16 patients with diagnostically proven UC were evaluated for SP receptor expression. The CD and UC specimens were subdivided into groups whose final pathological diagnosis was either positive or negative for CD or UC. Thirteen patients with CD had pathological confirmation of CD, whereas 6 patients either had uninvolved regions or did not have any pathological evidence of CD in regions of resected bowel. Eleven patients with UC underwent a colectomy and were pathologically positive for UC. Eight patients with UC subsequently had an ileostomy takedown procedure and had pathologically normal specimens. Two asymptomatic patients with a previous diagnosis of UC had a colectomy performed to avoid the risk of carcinoma; in both specimens, the pathological diagnosis was quiescent UC. Several of the patients with CD and UC are included in both pathologically positive and negative groups because tissue was obtained during the primary (e.g., colectomy) and reanastomotic (e.g., ileostomy takedown) procedures. The control specimens included six colon specimens, four resected for carcinoma and two for diverticulitis, in which the tissue samples were taken at least 4 cm from the primary disease site. Addi- tionally, four ileostomy samples were obtained in which the primary colonic diseases before the ileostomy takedown procedure included one with Ogilvie's syndrome, two with familial polyposis, and one with colovaginal fistulas. A gastrointestinal pathologist determined the pathological diagnosis for all tissues without knowledge of SP receptor expression.

Preoperative medications of all patients were obtained. Ta- ble 2 lists the CD- and UC-positive patients taking either steroids or other anti-inflammatory drugs before surgery. Ste- roid use was defined as any oral, parenteral, or rectal supposi- tory administration 1 week before surgery. Other anti-inflammatory drugs included sulfasalazine (or its derivatives), azathioprine, or metronidazole. Nine of the 12 CD-positive patients and 7 of the 11 UC-positive patients were taking steroids preoperatively. One of the CD-negative patients and none of the UC-negative patients was taking steroids before surgery was performed. None of the control patients was receiving either steroids or anti-inflammatory agents before surgery was performed.

T issue Preparat ion

Quantitative radioligand binding for the SP receptor was performed as previously described. 24 Briefly, surgically resected specimens from patients with CD, patients with UC, or control patients were obtained immediately after surgical removal. Once removed, the specimens were rapidly placed in a plastic mold, embedded in Tissue Tek O.C.T. compound (Miles, Elkhart, IN), and frozen on dry ice. The tissue was then stored at -80°C until sectioning. Specimens were sectioned at 20 t.tm using a Hacker-Bright microtome cryostat (Hacker Instruments, Fairfield, NJ) at -20°C, thaw mounted on Su- perfrost/Plus glass slides (Fisher Scientific, Pittsburgh, PA),

852 MANTYH ET AL. GASTROENTEROLOGY Vol. 109, No. 3

Table 1. Number of Patients Categorized by Sex, Age, and Specimen Site for Those Pathologically Positive for CD, Pathologically Positive for UC, Pathologically Negative for CD, Pathologically Negative for UC, and Controls

CD-positive CD-negative UC-positive UC-negative Control

Sex Male 3 2 5 4 9 Female 10 4 6 6 2

Mean age (SEM) 36.0 (3.3) 37.2 (6.1) 33.3 (4.2) 37.6 (2.8) 49.0 (4.8) Specimen site

Ileum 6 2 - - 1 - - Ileum and colon 2 . . . . Colon 4 1 11 2 6 Ileostomy 1 3 - - 7 5

and stored desiccated at - 80°C until the day of receptor binding.

SP Radiolabeling and Receptor Binding Assay

125I-Bolton-Hunter SP was synthesized using a previously published protocol. 2v On the day of the experiment, the slide-mounted tissue sections were thawed to room temperature. The slides were preincubated in 50 mmol/L Tris-HCl containing 0.005% (vol/vol) polyethylenimine, pH 7.4, at room temperature for 10 minutes. The sections were then

Table 2. Effects of Steroids or Other Anti-Inflammatory Agents on SP Receptor Density in Lymphoid Aggregates of Pathologically Positive Patients With CD and UC and Pathologically Negative Patients With CD

SP receptor Pathology n density a P value ~

Preoperative steroids CD-positive 10 9327 _+ 276 NS

CD-negative 2 8025 __ 18 UC-positive 8 9801 + 253

No steroids CD-positive 3 9321 _+ 601 NS CD-negative 4 9254 __ 289 UC-positive 3 9272 _+ 127

Other anti- infiammatories CD-positive 3 9089 _+ 519 NS

CD-negative 2 9205 __+ 276 UC-positive 4 9346 __ 167

NOTE. Patients were divided into groups that had been administered steroids (orally, intravenously, or rectally) within 7 days of their opera- tion (preoperative steroids), no steroids administered within 7 days of surgery (no steroids), or other anti-inflammatory agents within 7 days of surgery (other anti-inflammatories). Other anti-inflammatory medications included sulfasalazine or its derivatives, azathioprine, or metronidazole. A one-way analysis of variance of the densitometric values of the lymphoid aggregates in CD- or UC-positive patients showed no significant difference in SP receptor density among the groups. Similar not significant values were obtained among the three groups in SP receptor density in blood vessels (data not shown). aValues are expressed as mean _+ SEM. ~P > 0.10.

incubated at room temperature for 1 hour in a solution containing 100 pmol/L 125I-Bolton-Hunter SP containing 50 mmol/L Tris-HC1, 3 mmol/L MnCl2, 200 mg/L bovine serum albumin, 2 mg/L chymostatin, 4 mg/L leupeptin, and 40 mg/ L bacitracin; 1.5 mL of incubation solution was used for each tissue section. After incubation with the radioligand, the incubation medium was aspirated to waste, and the slides were rinsed four times for 4 minutes each in 50 mmol/L Tris-HCl, pH 7.4, at 4°C and then dipped twice in ice-cold, double- distilled water for 5 seconds each to remove salts. The slides were dried at 4°C under a stream of cold air and stored desiccated overnight. The slides were then placed in apposition to high-resolution x-ray film (Hyperfilm-Betamax; Amersham, Arlington Heights, IL) for 1 week. An 125I-microscale standard (Amersham) (20-~tm thick) with increasing concentrations of '2~I was also exposed to the x-ray film with the labeled slides. After exposure to the film, the slides were fixed in 4% formal- dehyde for 10 minutes at room temperature and stained with H&E. Autoradiograms were quantitated by densitometric analysis using the Bioquant Meg IV system (R&M Biometrics, Nashville, TN) with a solid-state camera (Dage-MTI Inc., Michigan City, MI) mounted on a Wild M5A stereomicroscope (Wild Leitz, Heerbrugg, Switzerland) with a light-field base. The autoradiographic image was displayed on a video monitor, and anatomic areas to be analyzed were outlined using a mouse and a digitizing pad (SummaSketch Plus; Summagraphics, Fairfield, CT). Densitometric analysis of the microscales was used to correct for the nonlinear response of the film as well as to compare different assays. Nonspecific binding (in the presence of 1 gtmol/L unlabeled SP) was negligible in all cases. Complete displacement of lzSI-SP by CP-96,345 (a nonpeptide SP receptor antagonist) established that the SP binding sites were authentic neurokinin 1 tachykinin receptors, e6 The concentrations of SP receptors for all groups are shown in Table

3.

Statistical Analysis

A minimum of six densitometric values were obtained from the autoradiogram for each tissue area, and these numbers were then averaged. The averaged densitometric values from each group of patients (either CD-positive, CD-negative, UC-


Table 3. SP Receptor Concentrations Expressed in Neurons, Blood Vessels, Lymphoid Aggregates, and Circular Muscle

Pathology Neurons Blood vessels Lymphoid aggregates Circular muscle

CD-positive 8646 (6817-10,216) 8047 (4016-9980) 9326 (7779-10,466) 4240 (1631-8365) CD-negative 8537 (7667-9537) 7903 (6316-9482) 8864 (8067-9950) 4236 (2829-5975) UC-positive 0 8775 (6427-10,068) 9633 (8565-10,883) 4246 (1329-6306) UC-negative 0 0 834 (0-2236) 2966 (1070-8447) Control 0 0 0 4336 (1034-7593)

NOTE. Values are expressed as the mean. Ranges of values are shown in ~arentheses (n = 6-13). All values are expressed as disintegrations per minute per milligram.

positive, UC-negative, or controls) were then pooled, and the SEM was calculated. To compare the differences in receptor densities among various pathological groups, an unpaired t test was performed to determine significance among the patient groups (i.e., CD-positive vs. UC-positive vs. controls) as well as receptor areas (i.e., neurons vs. lymphoid aggregates).

Results Pathologically CD-Positive Versus UC-Positive Specimens

The differential expression of SP binding between a representative tissue specimen from a pathologically positive CD specimen, pathologically positive UC specimen, or control specimen of uninvolved colon taken from a patient with adenocarcinoma is shown in Figure 1. A moderate level of 125I-SP binding is observed in the circular muscle of all tissues from patients with CD, patients with UC, and control patients. In the CD- and UC- positive specimens, very high concentrations of SP receptors were observed in submucosal and subserosal lymphoid aggregates. Small blood vessels scattered throughout the submucosa, muscularis, and serosal fat also contained ectopically expressed SP receptors in both the CD- and UC-positive patients. The CD-positive specimen also showed a high concentration of binding sites in enteric neurons in the myenteric plexus and the muscularis layer. The receptor distribution of the enteric neurons was discontinuous in nature, with large neurons and their respective arborizations containing receptors observed every few millimeters. Although the neurons expressing SP receptors were unevenly distributed in a tissue section of bowel, they could be identified in all CD-positive tissue. In contrast, although enteric neurons could be readily observed on H&E-stained sections of UC-positive and control tissue, no saturable binding of 125I-SP was found in neurons on any of the corresponding autoradiograms. SP receptor concentrations (expressed in disintegrations per minute per milligram tissue equiva- lents) in pathologically positive specimens from CD-posi- rive, UC-positive, and control patients are shown in Fig- ure 2. A large and statistically significant (P < 0.001)

increase in receptor density was noted in the small blood vessels and lymphoid aggregates in both the CD- and UC-positive patients when compared with control specimens. However, the enteric neurons of the CD-positive patients also showed a statistically significant (P < 0.001) increase in SP binding in comparison with the UC-positive patients and control patients. No difference in receptor concentration was found in the circular muscle in the CD-positive, UC-positive, and control groups (P > 0.1).

Pathologically CD-Negative Versus UC-Negative Specimens

Six patients with a history of CD underwent bowel resection and had negative pathological findings for CD. Three patients had ileostomy takedown after previous colonic resection for CD. One patient had previously undergone multiple small and large bowel resec- tions and finally had a proctectomy performed after numerous coloenteric and colocutaneous fistulas. Two patients underwent removal of the terminal ileum and proximal colon; CD pathology was confined exclusively to the colon. In these 2 patients, both the terminal ileum and proximal colon were assayed for SP receptor expression. In all 6 cases, the tissue that was processed for quantitative autoradiography was pathologically negative for any colitis. The pattern of SP receptor binding in the 6 cases of CD tissue was nearly identical to the active CD- positive tissues. As shown in Figure 3A and B, lymphoid aggregates, small blood vessels, and enteric neurons express a high concentration of SP receptors similar to that observed in CD-positive tissue. The receptors on the enteric neurons on CD-negative tissues were again expressed discontinuously throughout the myenteric plexus and muscularis.

Ten patients with a previous pathological diagnosis of UC subsequently underwent further surgical therapy. Seven patients had an ileostomy takedown, 1 had recur- rent fistulas and bleeding after an ileoanal pull-through and required a Brooke ileostomy (where ileal tissue was obtained), and 2 who were asymptomatic for UC at sur-


Figure 1. A comparison of SP receptors in CD-positive, UC-positive, and control tissue. A, D, and G are bright-field photomicrographs of sections of resected bowel stained with H&E. B, E, and H represent an autoradiogram of the Hyperfilm that overlaid the tissue sections shown in A, D, and G, respectively. The sections were incubated with 100 pmol/L of ~251-SP and represent total binding (TB). C, G, and / represent serial sections adjacent to and treated identically to the corresponding total binding sections except for the addition of 1 #mol /L of unlabeled SP and therefore represent nonspecific binding (NSB). The CD-positive tissue was obtained from the ileum of a patient who underwent resection for disease that was refractive to medical management. The H&E-stained section in A shows extensive lymphocytic infiltration throughout the submucosa and to the serosa. (B) Saturable 1251-SP binding is found in the lymphoid aggregates (LA) in the submucosal and serosal regions, small blood vessels (BY) in the submucosa, circular muscle (CM), and myenteric plexus (arrowheads). (D-F) The UC-positive specimen was taken from the colon of a patient with medically intractable disease who underwent a colectomy and ileoanal pull-through. SP receptors are localized to the LA, BV, and CM. Note the lack of binding to the myenteric neurons (D, arrowheads). The control specimen was obtained from a patient with carcinoma who had a subtotal colectomy. Binding in the control specimen is confined only to the CM (bars: A, 1.00 mm; D, 1.01 mm; G, 1.08 ram).

gery had a colectomy with an ileoanal pull-through performed to avoid the risk of carcinoma. In the latter two cases, the pathological diagnosis was quiescent UC. All UC tissue from the pathologically normal ileostomy tissue and the two cases of quiescent UC showed no ectopic SP receptors. An example of a UC ileostomy takedown sample in which the only discernable SP receptors are found in the circular muscle is shown in Figure 3C and D. No receptor up-regulation was found in small submucosal blood vessels or lymphoid aggregates. The same pattern of receptor binding was found in the circular

muscle of the control ileostomy tissue (Figure 2D and E).

The increase in SP binding sites in pathologically negative patients with CD on the enteric neurons, small blood vessels, and lymphoid aggregates in comparison with pathologically negative patients with UC or control patients is shown in Figure 4. A highly significant (P < 0.001) difference in SP receptors was found between the CD-negative tissue and the UC-negative and control tissue in enteric neurons, blood vessels, and lymphoid aggregates. However, the differences between the circular


qV

10000

8000

6000

4000

2000

N BV LA

_ T

\ \ \ \ \ \ \ \ \ \

CM

Rgure 2. Graphic representation of the total SP receptor density of CD-positive (B) (n = 13), UC-positive (r-I) (n = 11), and control tissues ([ ]) (n = 11). The four regions analyzed in each group were enteric neurons (N), small blood vessels (BY), lymphoid aggregates (LA), and circular muscle (CM). The receptor density is expressed as disintegrations per minute per milligram tissue obtained from comparing tissue samples with radioactive microscale standards containing known amounts of 1251 cut at 20 Ilm. Error bars represent the SEM of all of the collected samples. Note that CD-positive tissue had a highly significant (P < 0.001) increase in SP receptor density in the enteric neurons in comparison with UC-positive and control tissue. Significant differences in binding were noted in CD- and UC-positive tissues and control specimens (P < 0.001) in the blood vessels and lymphoid aggregates, but no significant differences (P > 0.10) were found between CD- and UC-positive tissue in these areas. The circular muscle had nearly identical levels of SP receptor density in the CD-positive, UC-positive, and control tissues (P > 0.10).

muscle of the CD-negative, UC-negative, and control circular muscle were not significant (P > 0.10). No difference in receptor density was noted between CD-posi- rive and CD-negative specimens in any region (P > 0.10).

Preoperative Steroid and Anti-inflammatory Medications

SP receptor concentrations of lymphoid aggregates of patients with CD and UC either receiving or not receiving preoperative steroids or other anti-inflammatory medications are shown in Table 2. Nine of 12 CD-positive patients and 8 of 11 UC-positive patients were administered steroids preoperatively. No significant change in receptor density on the lymphoid aggregates was found between the CD and UC group that received preoperative steroids when compared with those that did not. Three of 12 CD-positive and 4 of 11 UC-positive

patients had received other anti-inflammatory drugs; again, no significant change in receptor density was noted when compared with patients not receiving these drugs.

Discussion

In this study, we have used quantitative receptor autoradiography to compare the SP receptor density and distribution on surgical specimens from patients with CD and UC that were either pathologically positive or negative for colitis. Several significant findings are re- ported. In all CD tissue examined, a high concentration of SP receptors was localized on lymphoid aggregates and small blood vessels and in enteric neurons in the myenteric plexus and throughout the muscularis. In contrast, all pathologically positive UC tissue examined expressed high concentrations of SP receptors on lymphoid aggregates and small blood vessels but showed minimal binding on neuronal tissue.

The differential expression of SP receptors in CD vs. UC may aid in the diagnosis of patients who do not fit clinically or pathologically into one disease state. Ap- proximately 5 %-15 % of patients are diagnosed with indeterminate colitis, a term used when no reliable diagnostic feature categorizes a patient. 4 Although no clinical study has examined the rate of misdiagnosis of CD and UC, it is well known among gastroenterologists and sur- geons that a significant population of patients with IBD will have a change in their diagnosis. It is currently estimated that approximately 30%-50% of patients with CD will have disease in the colon. 28'29 Incorrect diagnosis of IBD in these patients could lead to improper medical and surgical therapy. Particularly hazardous in a patient with CD is a colectomy with construction of an ileal pouch reservoir because a significant number of these patients will have a recurrence of CD in the pouch, leading to chronic inflammation and pouch failure. 7'3° Al- though we found that all patients with CD examined show continual ectopic SP receptor expression and have a unique receptor distribution in comparison with UC, the potential for diagnostic assistance is still problematic. In our assay, the enteric neurons expressing the SP receptors in CD are located in the muscularis and require a full-thickness sample of bowel, obviating endoscopic biopsy sampling. Thus, this test may be of greater use in examination of resected bowel from patients with IBD in which traditional pathological distinctions between CD and UC are not found or in full-thickness rectal biopsy specimens.

In addition to CD having a unique distribution of SP receptors in comparison with UC, CD tissue, unlike UC specimens, continued to express high levels of SP recep-

D

Figure 3. Comparisons of SP receptor binding among CD-negative, UC-negative, and control tissue. A, C, and Eare bright-field photomicrographs of the H&E-stained tissue sections, whereas B, D, and F are the corresponding autoradiograms that overlaid the tissue sections. (A and B) The CD-negative specimen was taken from the ileostomy of a patient who 10 weeks previous to the takedown procedure had a resection of the terminal ileum and proximal colectomy for CD. At the time of the ileostomy takedown, the patient was asymptomatic and the final pathological analysis was negative for CD. Similar to the CD-positive tissue seen in Figure 1, SP receptor up-regulation is observed on the lymphoid aggregates (LA) in the serosal region, small blood vessels (BY) in the submucosa, circular muscle (CM), and the myenteric plexus and its arborizations (arrowheads). (C and D) The UC-negative tissue was obtained from a patient undergoing a complete colectomy for carcinoma avoidance. The patient was asymptomatic at the time of surgery, and the pathological diagnosis was quiescent UC. Only a moderate amount of binding is observed in the circular muscle with no detectable receptors in the lymphoid aggregates, small blood vessels, or enteric neurons. (E and F) The control tissue was taken from the ileostomy of a patient with familial polyposis coli who had previously undergone a colectomy and was now undergoing a reanastomotic procedure. The patient was asymptomatic, and pathological analysis showed only normal ileum. SP receptors are present only on the circular muscle of the control tissue (bars: A, 0.89 mm; C, 0.90 mm; E, 0.83 mm).


1 0 0 0 0

8 0 0 0

1~ 6 0 0 0

4 0 0 0

2 0 0 0

N BV LA CM

Figure 4. SP receptor density of pathologically CD-negative ( I ) (n = 6), UC-negative ([q) (n = 10), and control t issue (i~) (n = 11). Highly significant elevation of SP receptor binding (P < 0.001) was noted between CD-negative tissue in enteric neurons (N), small blood vessels (BY), and lymphoid aggregates (LA) when compared with the same regions in UC-negative and control tissue. No significant differences in receptor density (P > 0.10) were found between CD-negative, UC- negative, and control circular muscle. CM, circular muscle.

tors on enteric neurons, lymphoid aggregates, and small blood vessels on both pathologically positive and negative surgical specimens, In asymptomatic patients with CD undergoing reanastomotic procedures after resection of an area of active colitis or in grossly and pathologically normal tissue, SP receptors continued to be expressed at high levels in a pattern identical to clinically active, pathologically positive CD tissue. Patients with UC showed no increase in SP receptors in either quiescent colitis tissue taken from colonic resection or from ileal tissue (which was pathologically negative for UC) obtained from reanastomotic procedures. In these tissues, SP receptors were found only in the normally (similar to control patients) expressed circular muscle.

The observation that SP receptors are found in the small blood vessels, lymphocytic aggregations, and enteric neurons in pathologically normal regions of bowel in patients with CD may offer a rational explanation to the random skip lesions that are a hallmark of this disease. Wi th the persistent ectopic expression of the SP receptor in pathologically normal CD tissue, a perturba- tion in the vasculature, immune, or nervous systems in the bowel of patients with CD could lead to the release of SP, which activates the up-regulated receptors causing

inflammation, pain, and vascular leakiness. Previous re-

ports have indicated that SP levels are increased in patients with active UC, 3~ but peptide levels in quiescent UC or CD were not examined. Therefore, gut inflammation may proceed in an arbitrary manner anywhere in the alimentary tract if the continually high levels of SP receptors are stimulated. The lack of up-regulation of SP

receptors in quiescent UC and in ileostomy tissue sug- gests that SP contributes to the inflammatory response in UC only in the colon (and occasionally the ileum) and only during an acute exacerbation of the disease.

The differential expression of SP receptors between CD and UC is independent of steroid and other anti- inflammatory drug usage. A criticism that is often cited when comparisons of inflammatory mediators are made between patients with IBD is the effects of medications. In particular, steroids have been shown to decrease pro- duction of several cytokines and to decrease messenger

RNA for the SP receptor in vitro and in vivo.~2-~ How- ever, in this study, the changes in SP receptor concentrations seem to be independent of the preoperative steroid or other anti-inflammatory status in either patients with

CD or UC (Table 2).

The ectopic expression of SP receptors may provide insight into the pathogenesis of inflammation in CD and UC. Several lines of evidence implicate SP as a major contributor to the inflammatory response. Activation of primary, small diameter, unmyelinated afferent neurons

causes release of SP from nerve endings, causing vasodila- tion and plasma extravasation) 6 Because of the central role that SP plays in mediating this neurogenic inflammation, several investigators have studied the effects of the neuropeptide on the immune system and the vasculature. SP has been shown to have numerous effects on immune cells, including mast cell degranulation leading to an augmentation of vascular permeability~7'~s; release of interleukin 1, interleukin 6, and tumor necrosis factor from human monocytes~9; and enhancement of neutrophil cytotoxic effects. *° In the vascular system, SP receptors are found on endothelial cells, 4~ and treatment with SP causes expression of endothelial leukocyte adhesion molecule 1 on cultured endothelial cells 42 and increased adhesion of polymorphonuclear cells to cultured bronchial epithelial cells. 43 Furthermore, stimulation of SP-

containing afferent nerves leads to plasma extravasation, which can be inhibited by SP receptor antagonists. <'* Because of the strong evidence that SP may modulate inflammation, SP has been implicated in propagating the inflammatory activity in such diverse diseases as arthritis, ~5 asthma, 46 neural . .47 IBD -'~'J'~ degeneration, and .

The significance of the apparent up-regulation of SP

858 MANTYH El" AL. GASTROENTEROLOGY Vol. 109, No. 3

receptors in the enteric neurons of patients with CD but not patients with UC is not clear; however, recent work may be insightful. Early studies have shown hyperplasia of ganglion cells in the submucosal or myenteric plexuses in CD, 49'5° and the presence of these enlarged neurons is now generally accepted as a pathological feature of CD. In addition, electron microscopy of CD tissue has shown extensive axonal degeneration and infiltration of the gut plexuses with mast cells, lymphocytes, and plasma cells.5~ Other investigators have examined alterations in motor activity in animal models of gut inflammation and have suggested that the motility changes are caused by pertur- bations in inflammatory mediators that subsequently in- fluence enteric neuron and epithelial function. 52 How- ever, it is not known if the increase in SP receptor density represents either ectopic expression, up-regulation, mi- gration of SP receptor-containing cells (in blood vessels or lymphoid aggregates), or a concentration of receptors after activation and endocytosis of the receptor. At least for neurons, we have recent evidence to indicate the latter. We have shown that after endogenous SP release in either the spinal cord (stimulated by capsaicin or a pain- ful stimulus) or the ileum (by Clostridium difficile toxin A injection), the SP receptor is rapidly endocytosed and dramatic morphological changes occur in the cellular architecture. 53'54 In the ileum, the enteric SP receptor- expressing neurons appear swollen with thickened axons and dendrites. Therefore, it is conceivable that the increase in receptor density in CD may reflect the cellular concentration of SP receptors and subsequent morphological changes after a toxin- or chemically induced SP release. The relationship of these observations to IBD must remain speculative until reproducible animal models of chronic relapsing enterocolitis are examined for SP receptor changes.

Finally, the differential expression of SP receptors in CD and UC may have therapeutic implications. Recently, several specific, nonpeptide antagonists to the SP receptor have been developed. 25 These antagonists have been shown to block SP receptor activation both in vitro and in vivo. 55 Specifically, the antagonists inhibit SP-induced neurogenic inflammation, such as plasma extravasation in the lung 56'57 and plasma leakage in the knee joint, 25 as well as specific spinal pain pathways. 58 In the gut, the SP receptor antagonist CP-96,345 effectively blocked granuloma formation in a Trichinella spiralis mouse model of inflammation. 59 In addition, the same SP receptor antagonist inhibited the histological and physiological effects of C. difficile toxin A-induced enterocolitis in the rat, 6° suggesting that the SP receptor modulates these inflammatory responses. Finally, our group has recently

shown that several of the nonpeptide SP receptor antagonists effectively eliminate SP binding to CD and UC tissue at nanomolar concentrations. 26 Therefore, the nonpeptide receptor antagonists are novel compounds that may potentially ameliorate the proinflammatory effects of SP in IBD and other inflammatory conditions such as asthma and arthritis. Based on our study, we hypothesize that SP receptor blockade may be beneficial in active (and presumably pathologically positive) CD and UC. Additionally, the antagonists may also be useful in quiescent (and presumably pathologically negative) CD in which the continual up-regulation of SP receptors may indicate a subclinical disease state.

In conclusion, we have shown the differential distribution of SP receptors in CD and UC. SP receptors are ectopically expressed in lymphoid aggregates, small blood vessels, and enteric neurons of CD in both active, pathologically positive surgical specimens as well as in quiescent, pathologically negative samples. In UC, the SP receptor up-regulation is confined to the blood vessels and lymphoid aggregates of active, pathologically positive specimens. No SP receptor up-regulation was evident on quiescent, pathologically negative UC tissue. These findings may have significant diagnostic, etiologic, and therapeutic importance in IBD.

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Received February 6, 1995. Accepted April 19, 1995. Address requests for reprints to: Christopher R. Mantyh, M.D.,

Department of Surgery, Duke University Medical Center, Box 3614, Durham, North Carolina 27710. Fax: (919) 286-5530.

Supported by Veterans Affairs grant 0010 and National Institutes of Health grant GM-15904.

The authors thank Evelyn Stimson and Kelly Van Koughnet for excellent technical support.

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Differential expression of substance P receptors in patients with Crohn's disease and ulcerative colitis