Production of immunoglobulin G and G1 antibodies to cytoskeletal protein by lamina propria cells in ulcerative colitis

GASTROENTEROLOGY1995;109:3-12

ALIMENTARY TRACT Production of Immunoglobulin G and G1 Antibodies to Cytoskeletal Protein by Lamina Propria Cells in Ulcerative Colitis LIVIA BIANCONE,* ADITYA MANDAL,* HOYEOL YANG, § TANYA DASGUPTA, t ALESSANDRO OMERO PAOLUZI,* ADRIANA MARCHEGGIANO,* PAOLO PAOLUZI,* FRANCESCO PALLONE, li and KIRON MOY DAS* *Cattedra di Gastroenterologia,UniversitaLa Sapienza,Rome, Italy; "Departmentdi MedicinaSperimentaie,Universita'di ReggioCalabria, Catanzaro, Italy; tDivisionof Gastroenterology,RobertWoodJohnsonMedicalSchool, New Brunswick,New Jersey;and §Departmentof Medicine, JerseyCity MedicalCenter, New Jersey

See editorial on page 307. Background & A i m s : Recent studies suggest an autoantigenic role for tropomyosin-related protein(s) in ulcerative colitis (UC). This study examined whether immunoglobulin G and G1 subclass antibodies against tropomyosins are produced spontaneously by the lamina propria mononuclear cells (LPMCs) that infiltrate the inflamed UC tissue. Methods: LPMCs were isolated from colonic biopsy specimens from 29 patients with UC, 15 with colonic Crohn's disease (CD), and 13 with non-inflammatory bowel disease (IBD). The autologous peripheral blood mononuclear cells (PBMCs) were obtained. Cells were cultured in vitro and unstimulated for 10 days. Spontaneous production of immunoglobulin G and G1 antibodies against tropomyosins was measured by enzyme-linked immunosorbent assays using highly enriched tropomyosins from skeletal muscle and colonic mucosa. Results: The total immunoglobulin G produced by LPMCs from both patients with UC and CD was comparable but higher than from patients with non-IBD (P < 0.05). However, immunoglobulin G antibodies to tropomyosins were higher in patients with UC than in patients with CD (P < 0.04) and non-IBD (P < 0.02). LPMCs from patients with symptomatic UC produced higher immunoglobulin G antibodies to tropomyosins than patients with UC in remission (P < 0.03), symptomatic CD (P < 0.04), and non-IBD (P < 0.02). Immunoglobulin G antibodies to tropomyosins predominantly belonged to immunoglobulin G1 subclass. The autologous PBMCs showed comparable results. Conclusions: Immunoglobulin G antibodies predominantly belonging to immunoglobulin G1 subclass and reactive against tropomyosin-related protein(s) are spontaneously produced by LPMCs from the colonic mucosa in patients with UC.

everal observations indicate that autoimmune phenomena are involved in the pathogenesis of ulcerative colitis (UC). ~-4 In patients with UC, a colon tissue-bound immunoglobulin (Ig) G antibody, termed colitis-colonassociated IgG, was found to recognize a colonic protein (P40) with a relative molecular weight of 40,000. 5,6 Autologous colitis-colon-associated IgG also reacted with the P40. However, the P40 was not recognized by IgG eluted from the colon of patients with Crohn's disease (CD) colitis, diverticulitis, and other control IgG antibodies. 6 Sera from patients with UC also reacted with the Colon extract enriched in P40. v P40 recognized by the colitis-colon-associated IgG antibody was found in all colons but was absent in the ileum, duodenum, stomach, and liver, suggesting organ specificity of the antigen. 6 These studies indicate that P40 is a normal colonic protein and the colitis-colon-associated IgG antibody seems to be specific to u c and represents an autoantibody. Recently, the P40 was purified from the colon to homogeneity, partially sequenced after proteolytic digestions, and the two peptides derived therefrom showed 9 3 % - 1 0 0 % identity with the cytoskeletal protein tropomyosins (TMs). 8 TMs are cytoskeletal microtubular proteins present in all eukaryotic ceils with organ-specific isoforms. 9 Eighty-five percent of patients with UC had circulating autoantibodies against the TMs, whereas patients with CD and diarrhea caused by specific pathogens

S

Abbreviations used in this paper."ELISA,enzyme-linkedimmunosorbent assay; HBSS-CMF,ca!cium- and magnesium-freeHank's balanced salt solution; LPMC, lamina propria mononuclearcell; OD, optical density; PBMC, peripheralblood mononuclearcell; PWM, pokeweed mitogen;SDS, sodiumdodecylsulfate; TM, tropemyosin. © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00

4

BIANCONE ET AL.

and patients with systemic lupus erythematosus did not have them. 8 In this study, we examined whether antibodies reactive against TMs are actively produced by the lamina propria mononuclear cells (LPMCs) infiltrating the inflamed coIonic tissue. The I g G subclass distribution of the antibodies against TMs produced in vitro by LPMCs and also by autologous peripheral blood mononuclear cells (PBMCs) from patients with UC, CD, and n o n - i n flammatory bowel disease (IBD) controls was analyzed.

Materials and Methods Patients Fifty-seven patients undergoing colonoscopy were studied. There were 29 patients with UC (20 male and 9 female; mean age + SD, 46 +_ 14 years; range, 1 5 - 7 7 years); l0 of the 29 patients (7 male and 3 female) were symptomatic. The colitis was distal in 12 patients, left-sided in 13 patients, and extensive in 4 patients. At the time of the study, 6 patients were taking steroids, 15 patients were taking mesalamine medications (orally in 8 patients, rectally in 4, orally and by enema in 3), and 8 patients were not receiving any treatment. The mean (-+-SD) duration of the disease in patients with UC was 7 + 7 years (range, 0 . 5 - 2 6 years), 8 -+ 8 in the symptomatic group (range, 0 . 5 - 2 6 years), and 6 + 5 for patients in remission (range, 1 - 2 1 years). Patients, with non-UC included 15 patients with colonic CD (10 male and 5 female; mean age ___ SD, 40 -4- 13 years; range, 1 7 - 6 2 years) and 13 with nonIBD (7 male and 6 female; mean age + SD, 55 --- 15 years; range, 2 6 - 7 6 years). In the CD group, 10 patients were symptomatic, and 5 patients were in remission. Eight patients were taking steroids, and 5 were treated with oral mesalamine. The mean duration of the disease (+SD) was 4.8 _+ 3.6 years (range, 1 - 1 2 years). The non-IBD group included 3 patients with irritable bowel syndrome, 5 patients with diverticular disease, 3 patients with colonic polyps, and 2 patients with hemorrhoids. In all subjects, the diagnosis was made according to the usual clinical, endoscopic , radiological, and histological criteria. Disease activity was assessed in patients with UC by the clinical history and endoscopy findingsm '11 and in patients with CD by the Bristol's simple index, 12 supplemented by laboratory measurements. 13 The endoscopic activity of mucosal inflammation was assessed during colonoscopy according to the criteria described by Gomes et al. 14 on a 0 - 3 scale (0, normal; 1, remission; 2, mild or moderate disease; and 3, severe disease with ulcers and spontaneous bleeding). The morphology of the mucosal biopsy specimens was systematically analyzed by a single pathologist (A.M.) who was unaware of gross findings and the experimental data. The histological grading was assessed according to a 0 - 4 scale (0, normal mucosa; l, architectural distortion of crypts, mucosal atrophy, and no active inflammation in the lamina propria; 2, mild active inflammation in the lamina propria, some crypt abscesses, mild goblet cell depletion, and architectural changes; 3, moderate

GASTROENTEROLOGYVol. 109, No. I

active inflammation in the lamina propria, crypt abscesses, more evident goblet cell depletion, superficial erosions, and architectural changes; and 4, severe active inflammation in the lamina propria and significant epithelial destruction with ulcerations.

Biopsy Samples In patients with IBD, 3 - 5 biopsy samples were obtained during colonoscopy for isolation of LPMCs. Two more biopsy specimens were obtained for the histological evaluation of inflammation. In the UC group, biopsy specimens were obtained from the distal 20 cm of the colon (rectum and sigmoid colon) in 21 patients and from the proximal to the sigmoid colon in 8 patients. In the CD group, biopsy specimens were obtained from the distal 20 cm of the rectum and sigmoid colon in 4 patients and from the colon proximal to the sigmoid colon in 11 patientsl In non-IBD controls, biopsy specimens were obtained from endoscopically and histologically normal mucosa (grade 0) from the rectum and sigmoid colon (11 patients) and from the descending colon (2 patients).

Blood Samples Heparinized venous blood samples were obtained from each patient during the visit for colonoscopy for the isolation of PBMCs.

Isolation of LPMC LPMCs were isolated under sterile conditions using the ethylenediaminetetraacetic acid (EDTA)-collagenase sequence 15 with modifications for small samples as described previously. 16 Briefly, biopsy specimens were placed in a glass tube containing a complete medium (RPMI 1640 + 10% fetal calf serum, 25 mmol/L HEPES buffer, 2 mmol/L L-glutamine, 100 ~g/mL streptomycin, 100 U/mL penicillin, and 50 gg/ mL gentamicin) (Flow Laboratories, England) for 5 minutes at 25°C and then were placed in a Petri dish and minced. To remove epithelial cells , samples were incubated for l0 minutes at 37°C in calcium- and magnesium-free Hank's balanced salt solution (HBSS-CMF) containing 0.75 mmol/L EDTA and 10 mmol/L HEPES buffer. After two washes, samples were incubated for 2 hours at 37°C in 5% CO2 in a complete medium containing 25 UlmL purified Collagenase (type V; Sigma Chemical Co., St. Louis, MO). The cells were collected and washed three times in HBSS, and the resulting LPMCs were resuspended in a 40% solution of Percoll (Pharmacia, Uppsala, Sweden). An isotonic solution of Percoll, consisting of nine parts Percoll and one part 10X HBSS-CMF (pH 7.4, 290 mOsm) was used to prepare dilutions with HBSS-CMF. In a glass tube, 2 mL each of 100%, 60%, 40%, and 30% Percoll were layered. The tube was centrifuged at 400g for 25 minutes, and the cells at the interface of the 6 0 % - 4 0 % Percoll layers (containing LPMCs) were collected. LPMCs were washed, counted, and the viability assessed by 0.1% trypan blue exclusion. Contamination by epithelial cells of the LPMCs suspension ranged between 10% and 16%. LPMC yield ranged

July 1995

from 500,000 to 3,500,000 for 3 - 5 biopsy specimens (mean _+ SD, 8 X 106 _+ 1.5 X 106 LPMC/g wet tissue; range, 2 × 106 to 14 X 106 LPMC/g). The viability of LPMCs ranged from 80% to 90% before cultures and from 60% to 75% on the 10th day of culture. The autologous PBMCs were obtained from heparinized venous blood layered on a Fycoll-Hypaque (Pharmacia) density gradient. The viability of PBMCs ranged from 90% to 95% before cultures and 80% to 90% on the 10th day.

LPMC and PBMC Cultures Ceils were resuspended in a complete medium (2 X 10 ~ per 2 mL), placed in 2.8 mL wells of tissue culture plates (Falcon Plastic, Franklin Lakes, NJ), and cultured unstimulated at 37°C in 5% CO2. Paired cultures were run as controls, providing the wells with 1 Hg/well pokeweed mitogen (PWM; Gibco Laboratories, Grand Island, NY). On the 10th day, ceils were harvested, counted, and checked for viability by 0.1% trypan blue exclusion. Supernatants were collected, frozen within 1 hour, and stored at - 2 0 ° C until tested.

Purification of TMs TMs were purified from human skeletal muscle and colonic mucosa following previously published methods 17'1. with minor modifications. Briefly, human muscle was obtained from an amputated leg, and normal colonic mucosa was obtained by stripping off the mucosa at the submucosal piane from fresh operative specimens of the colon. Tissue specimens were homogenized separately, and the solubilized proteins were subjected to heat treatment and centrifuged, and the heatresistant supernatant was further fractionated by ammonium sulfate (initially 45% saturation followed by 56% saturation). The 56% saturated ammonium sulfate precipitate was dissolved in water and dialyzed extensively against 10 mmol/ L imidazole (pH 7.0) containing 1 mol/L KC1, 2 mmol/L dithiothreitol, and 0.2 mmol/L phenylmethylsulfonyl fluoride. The concentration of the proteins was measured by a protein assay (Bio-Rad Laboratories, Hercules, CA) and analyzed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the immunoreactivity was examined by an enzyme-linked immunosorbent assay (ELISA) and immunotransblot analysis <* using both monoclonal and polyclonal antibodies (Sigma Chemical Co.) to TMs.

Estimation of Total IgG in Culture Supernatants ELISA plates were coated with the F(ab')2 fragments of the goat anti-human IgG (H + L chain) (Cappel, OrganonTeknika Corp., West Chester, PA) and diluted 1:1000 in a carbonate buffer (pH 9.6) (100 HL). After overnight incubation at 4°C, the plates were washed three times with phosphatebuffered saline and 0.05% Tween 20 (Sigma Chemical Co.), and then supernatants (1:1) were added in triplicate (100 ~L/ well). As the standard, purified polyclonal human IgG (Cappel Laboratories, Durham, NC) in 12 serial dilutions was used.

AUTOANTIBODIES FROM MUCOSAL LYMPHOCYTES IN UC

5

After 2 hours at 25°C and overnight incubation at 4°C, plates were washed and then alkaline-phosphatase-conjugated goat anti-human IgG (Sigma Chemical Co.) was added (100 HE at 1:1000). After 1 hour at 25°C and three washings, the substrate p-nitrophenyl-phosphate-disodium was added (100 HL/well) (5 Hg/mL). After 1 hour at 25°C, plates were read at 405 nm in an ELISA reader. Data were expressed as nan0grams per milliliter and calculated from the standard curve by double logarithmic regression analysis.

Estimation of Total IgG1 in Culture Supernatants For the IgG subclass analysis, unstimulated supernatants from 20 of 29 patients with UC, 10 of 15 patients with CD, and 13 of 13 patients with non-IBD were available. The supernatants from the remaining patients were not sufficient for these analyses. Plates were coated with the F(ab')2 fragments of the goat anti-human IgG1 (clone SG-16; Sigma Chemical Co.) at 1:1000 in a carbonate buffer (100 HL/well). After being maintained overnight at 4°C and after three washes, supernatants were added as above. As the standard, purified polyclonal human IgG1 (Sigma Chemical Co.) in 12 serial dilutions was used. After 2 hours at 25°C and after being maintained overnight at 4°C, plates were washed, and the alkaline-phosphatase-conjugated goat anti-human IgG1 (clone GG-5; Sigma Chemical Co.) was added (1:1000). The reaction was developed as above, plates were read at 405 nm, and data were expressed as nanograms per milliliter.

Estimation of IgG and IgG Subclass Antibodies Against TMs in Culture Supernatants ELISA plates (Falcon Plastic) were coated with the enriched TMs (0.5 Hg/well) diluted in the carbonate buffer (pH 9.6) (100 HL/well). IgG antibodies to TMs were measured using the highly enriched TMs extracted from human skeletal muscle as antigen(s), whereas for IgG subclass determination, ELISAs were performed using the enriched TMs extracted from human colonic mucosa as antigen(s). As a blank, additional wells in both ELISAs were coated with bovine serum albumin (0.5 Hg/100 HL). After an overnight incubation at 4°C, plates were washed as described above three times in phosphatebuffered saline and 0.05% Tween 20 (200 HL/well). Nonspecific binding was blocked with 0.25% bovine serum albumin in phosphate-buffered saline (100 HE/well) for 30 minutes at 37°C. After three washings, diluted supernatants (1:1) were added in triplicates (100 btL/well). Plates were incubated for 2 hours at 25°C and then overnight at 4°C. After three washings, the alkaline-phosphatase-conjugated goat anti-human IgG (100 HL at 1:6000) (Sigma Chemical Co.) was added for the measurement of IgG antibodies to TMs. For IgG subclass determination, subclass-specific ELISAs were performed using the enriched TMs extracted from colonic mucosa as antigen(s). The method was the same as described above. However, the detection system included alkaline-phospharase-conjugated

6

BIANCONE ET AL.

GASTROENTEROLOGY Vol. 1 0 9 , No. 1

Table 1. Spontaneous Production of Total IgG by LPMCs and PBMCs From Patients With UC and CD and Non-IBD Controls Total IgG concentration

(ng/mL)

LPMCs Subject group UC Total Symptomatic Remission CD Total Symptomatic Remission Non-IBD controls Total

PBMCs

n

Median

Range

Median

Range

29 10 19

306 520 291

(90-823)" (234-823) b (90-811)

502 611 458

(137-899) (205-663) (137-899)

15 10 5

316 316 305

(106-986) ~ (106-986) (196-415)

496 536 374

(185-561) (185-561) (283-465)

13

136

(76-681)

313

(78-863)

"P < 0.05, all patients with UC vs. non-IBD. "P < 0.05, patients with symptomatic UC vs. patients with UC in remission and non-IBD. cp < 0.05, all patients with CD vs. non-IBD; no difference between UC and CD.

goat anti-human IgG1, IgG2, or IgG3 (Zymed, San Francisco, CA). The subclass-specific antibodies were added at 1:6000 (100 #L/well). After 1 hour at 25°C and three washings, the substrate p-nitrophenyl-phosphate-disodium was added (100 #L) (5 #g/mL). After 1 hour at 25°C, plates were read at 405 nm. Results for each sample were expressed as the mean optical density (OD) _+ SD of triplicate values with the blank value subtracted from each value. However, because OD levels were not normally distributed, when pooling values from different samples, data were expressed as the median OD and the range of the three OD values in each sample.

PBMCs cultured in vitro and unstimulated for 10 days. Total IgG levels were higher in LPMC supernatants from both patients with UC and CD compared with non-IBD controls (P < 0.05). Total IgG production by LPMCs did not differ in patients with UC and CD. Total IgG produced by LPMCs from patients with symptomatic UC was higher than from patients with UC in remission (P < 0.05) and controls with non-IBD (P < 0.05), whereas in patients with CD, the amount of total IgG was similar in both symptomatic and remission groups. Total IgG produced by PBMCs from the IBD group was higher than non-IBD controls; however, this was not statistically significant. PWM stimulation markedly increased the production of total IgG by PBMCs (unstimulated vs. stimulated, P < 0.04 for patients with IBD and P < 0.03 for patients with non-IBD) but not by LPMCs from all groups. The median values for total IgG (in nanograms per milliliter) produced by stimulated PBMCs were as follows: patients with UC, 766 (range, 473-987); patients with CD, 703 (range, 416-968); and patients with nonIBD, 681 (range, 539-903). The median values for total IgG (in nanograms per milliliter) produced by PWM-stimulated LPMCs were as follows: patients with UC, 348 (range, 145731); patients with CD, 331 (range, 295-867); and patients with non-IBD, 167 (range, 106-785).

Total IgG1 in Supernatants Table 2 shows the total IgG1 produced by unstimulated LPMCs and PBMCs from patients with IBD and non-IBD controls. LPMCs and PBMCs from all pa-

SDS-PAGE and Western Blot Analysis Enriched TMs extracted from human skeletal muscle and normal colonic mucosa were subjected to 10% SDS-PAGE. Immunoreactivity against polyclonal anti-TM antibody (Sigma Chemical Co.) was determined by Western blot analysis as described in our previous studies. 6'8

Table 2, Spontaneous Production of Total !gG1 by LPMCs and PBMCs From Patients With UC and CD and Non-IBD Controls Total IgG1 concentration LPMCs

Statistical Analysis Because values of total and TM-specific antibodies were not normally distributed in all groups, the nonparametrical Kruskal-Wallis test for small biological samples was used for the statistical analysis of the data. Therefore, results were expressed as the median and observed range in all tables and figures. The statistical dependence of the antibody production (OD) and the mucosal degree of inflammation were analyzed in all groups. The relation was considered in terms of linear trend and expressed by the variance explained by the regressi°n (R2). 19

Results Total IgG in Supernatants Table 1 shows the spontaneous production of total IgG in patients with IBD and controls by LPMCs and

(ng/mL)

Subject group UC Total Symptomatic Remission CD Total Symptomatic Remission Non-IBD controls Total

PBMCs

n

Median

Range

Median

Range

20 6 14

178 372 157

(49-553) ~ (149-553) ° (49-438)

510 510 437

(9-823) c (145-680) (9-823)

10 8 2

196 196 195

(41-843)" (41-843) (82-308)

295 536 159

(69-371) (185-561) o (69-250)

13

118

(50-456)

269

(51-838)

ap < 0.05, all patients with UC and CD vs. non-IBD; no significant difference between all patients with UC and CD. bp < 0.02, patients with symptomatic UC vs. UC in remission; P < 0.01, patients with symptomatic UC vs. non-IBD; P < 0.05, patients with symptomatic UC vs. symptomatic CD. °P < 0.05, all patients with UC vs. non-IBD and all patients with CD. Op< 0.05, patients with symptomatic CD vs. CD in remission.

July 1 9 9 5

MP

AUTOANTIBODIES FROM MUCOSAL LYMPHOCYTES IN UC

A

C

E

D

} 200K

!:i!,iiiiiii

--

116K-97K

i!!i'!i,,i~

{:~{!{

ili~iii~iiil

66K

!ii:iii i{ i~>{iii - - ~ 3 6 K 29K

46K

j

42K

3 8 K - -

ilii!i i

Figure 1. SDS-PAGE (10%) of highly enriched TMs extracted from human skeletal muscle (lanes A and B) and normal colonic mucosa (lanes C-E). Lanes A and C are stained with coomassie brilliant blue, and lane D is stained with silver nitrate. Lanes B and E show immunoreactivity against polyclonal antibody against TMs in Western blot analysis. Muscle TMs can be seen as doublets at relative molecular weights of 38K and 36K. The minor reactivity at higher molecular weight is probably caused by dimers from incomplete reduction. CoIonic mucosal TMs included several proteins at relative molecular weights of 46K, 42K, 4OK, 38K, and 32K. Lane MP contains marker proteins.

tients spontaneously produced detectable amounts of IgG1 after 10 days in culture. Total IgG1 levels produced by LPMCs were higher in both patients with UC and with CD than in non-IBD controls (P < 0.05). Total IgG1 produced by LPMCs did not differ between the whole group of patients with UC and CD. However, LPMCs from patients with symptomatic UC produced higher amounts of IgG1 than LPMCs from patients with symptomatic CD (P < 0.05), patients with non-IBD (P < 0.01), as well as patients with UC in remission (P < 0.02). Total IgG1 produced by PBMCs were higher in patients with UC than in patients with CD (P < 0.05) or non-IBD (P < 0.05). IgG Antibodies Against TMs in Culture Supernatants Figure 1 shows the highly enriched TMs purified from human skeletal muscle and colonic mucosa. The

muscle TMs appear as a doublet at relative molecular weights of 36K and 38K, and both bands reacted with the anti-TMs antibody as shown by Western blot analysis. Colonic mucosal TMs consist of several proteins at relative molecular weights of 46K, 42K, 40K, 38K, and 32K, and all of them reacted with anti-TM polyclonal antibody as shown in Figure 1 (lanes C-E). Western blot analysis using LPMC supernatants was negative in all groups, including the UC group. In the ELISA, using

7

the supernatants from cultured LPMCs against the enriched muscle TMs, the OD values are shown for all 57 patients in the scattergram (Figure 2). The median O D value for LPMCs from patients with UC was higher (OD, 0.153; range, 0.067-0.754) than that of either patients with non-IBD (OD, 0.108; range, 0.033-0.187; P < 0.02) or patients with CD (OD, 0.108; range, 0 . 0 8 2 0.158; P < 0.04). LPMCs from patients with symptomatic UC produced higher IgG antibodies to TMs (OD, 0.213; range, 0.091-0.754) than those from patients with UC in remission (OD, 0.127; range, 0.067-0.579) (P < 0.03), with symptomatic CD (OD, 0.104; range, 0.082-0.158; P < 0.04), and with non-IBD (P < 0.02). There were no differences between patients with symptomatic CD and CD in remission (OD, 0.114; range, 0.086-0.147). Results from the autologous PBMCs confirmed our previous findings, s showing that PBMCs from the total group of patients with UC produced higher IgG antibodies to TMs (OD, 0.266; range, 0.070-0.968) than controis with non-IBD (OD, 0.171; range, 0.099-0.282; P < 0.04), whereas the difference with the total group of patients with CD was not significant (OD, 0.207; range, 0.089-0.423). However, patients with symptomatic UC produced higher IgG antibodies to TMs than patients with symptomatic CD (P < 0.03), non-IBD controls (P < 0.02), as well as patients with UC in

1.0OO m .900 _

&

.800. .700 -~..6O0

--

•

A

& o .I

.41111 _

-~ . 3 0 0

--

0

.2~

--

.100

__

A

:

o

AA

• e~ •

-- - - ~ L P M C IsBMC L P M C PBMC ACTIVE iNACTiVE LILOERATI VE COLITIS

mum

-4L P M C PBMC TOTAL NON-IBO

LPMC PIMC LPMC PBMC ACTIVE INACTIVE CROHN'S DISEASE

Figure 2. Scattergram showing the OD values for the spontaneous production of IgG antibodies against TMs by LPMCs (e) and autologous PBMCs (A) from each of the 57 patients (29 with UC, 13 with non-IBD, and 15 with CD). LPMCs and PBMCs were cultured and unstimulated for 10 days, and supematants were tested against enriched TMs from human skeletal muscle by an ELISA. Each point indicates the OD value for each patient, and sofid lines are the median for individual groups. The median OD value for LPMCs in patients with UC was significantly higher when compared with non-IBD patients (P < 0.02) as well as with CD (P < 0.04). PBMCs from patients with active UC produced higher levels of IgG antibodies to TMs when compared with non-IBD controls (P < 0.02) and patients with symptomatic CD (P < 0.03) (nonparametrical KruskaI-Wallis Test).

8 BIANCONEET AL.

remission (P < 0.01). There were no differences between patients with symptomatic CD and patients with CD in remission and no differences between patients with CD and non-IBD patients. The duration and extent of IBD did not seem to influence the production of IgG antibodies to TMs by both LPMCs and PBMCs. In UC, patients treated with steroids had lower OD values (median, 0.097; range, 0.0670.579) compared with patients not treated with steroids (median, 0.154; range, 0.076-0.754). However, this difference did not reach statistical significance. The site from which the biopsy specimens were obtained seemed to influence the production of IgG antibodies to TMs by LPMCs from the diseased UC colon. LPMCs isolated from the distal large bowel (rectum and sigmoid colon) produced more antibodies to TMs (OD, 0.156; range, 0.076-0.754) than LPMCs isolated from the more proximal colon (OD, 0.096; range, 0.067-0.202) (P < 0.05). The site from which biopsy specimens were obtained did not affect the OD values in patients with CD (distal vs. proximal: OD, 0.146; range, 0.082-0.158 vs. OD, 0.100; range, 0.086-0.130) and non-IBD patients (distal vs. proximal: OD, 0.095; range, 0.033-0.187 vs. OD, 0.090; range, 0.082-0.098). PWM stimulation of LPMCs and PBMCs did not significantly increase the production of IgG antibodies to TMs when compared with unstimulated cultures. However, in patients with UC, PWM-stimulated LPMCs (compared with unstimulated LPMCs) produced more IgG antibodies to TMs (OD, 0.166; range, 0.035-970) than in patients with CD (OD, 0.101; range, 0.0830.193) (P < 0.05) and with non-IBD (OD, 0.093; range, 0.027-0.153) (P < 0.05). Stimulated LPMCs produced more IgG antibodies to TMs in patients with symptomatic UC than in patients with UC in remission (OD, 0.209; range, 0.056-0.970 vs. OD, 0.096; range, 0.035-0.722; P < 0.04), with symptomatic CD (OD, 0.095; range, 0.090-0.101; P < 0.04) and with nonIBD (P < 0.03).

IgG1 Antibodies Against TMs in Culture Supernatants The OD values for IgG1 antibodies to TMs were in the detectable range in 80% of the patients with IBD and only in 44% of controls with non-IBD. The median OD value for IgG1 antibodies to TMs, spontaneously produced by LPMCs in patients with UC, was 0.096 (range, 0.020-0.247), for patients with CD it was 0.065 (range, 0.005-0.192), and for controls with non-IBD it was 0.023 (range, 0.013-0.034). The production of IgG1 antibodies to TMs by LPMCs from patients with

GASTROENTEROLOGYVol. 109, No. 1

symptomatic UC was higher (0.105) than from patients with UC in remission (0.089; P < 0.05), with symptomatic CD (0.068; P < 0.05), and with non-IBD (P < 0.002). The ratio of IgG1 to TMs/total IgG1 in LPMC supernatants was significantly higher (P < 0.01) in patients with UC compared with patients with CD and with non-IBD. When the production of IgG1 antibodies to TMs was related with the total IgG antibodies to TMs produced by LPMCs, it was found that this ratio was higher in the total UC group (0.38) than in all of the patients with CD (0.16) (P < 0.05) and with non-IBD (0.02) (P < 0.03). The ratio of IgG1 to TMs/total IgG to TMs was even higher in patients with symptomatic UC (0.60) than in patients with UC in remission (0.38) (P < 0.05), with symptomatic CD (0.17) (P < 0.05), and with nonIBD (P < 0.01).

IgG2 and IgG3 Antibodies Against TMs in Culture Supernatants The spontaneous production by LPMCs of IgG2 and IgG3 antibodies to TMs in all the groups was undetectable in two thirds of the specimens. The OD values in the remaining patients were also too low for any statistical interpretation.

IgG and IgG1 Antibodies Against TMs and Endoscopic Assessment of Mucosal Inflammation in IBD: Comparison With Non-IBD Controls To evaluate whether IgG and tgG1 antibodies production against TMs is associated with the presence of active inflammation at the site from which LPMCs were isolated, we grouped patients with IBD according to Gomes' grades of < 2 (remission) and -->2 (active inflammation). As shown in Table 3, the IgG antibodies to TMs produced by LPMCs were higher in the actively inflamed colon of patients with UC when compared with patients with UC in remission (P < 0.04). This value in patients with UC was also higher than patients with CD with active inflammation (P < 0.03). When compared with non-IBD controls, LPMC production of IgG antibodies to TMs was higher in both the actively inflamed UC colon (P < 0.01) as well as in patients with UC with endoscopic remission (P < 0.02). The production of IgG1 antibodies to TMs by LPMCs in patients with UC with active endoscopic inflammation was higher when compared with the actively inflamed colon of patients with CD (P < 0.04) and the colon of patients with UC with endoscopic remission (P < 0.04) (Table 3). The IgG1 antibodies to TMs were lower in non-IBD

July 1995

AUTOANTIBODIES FROM MUCOSAL LYMPHOCYTES IN UC

9

Table 3. IgG and IgG1 Antibodies to TMs Produced by LPMCs From IBD Colon in Remission (Grade <2) and With Active Inflammation (Grades ->2) as Assessed by Endoscopy: Comparison With Non-IBD Controls IgG antibodies to TMs

Disease group

IgG1 antibodies to TMs

n

Median OD

Range

11 13

0.202 0.104

(0.078-0.754) a (0.067-0.279) °

8 5

0.086 0.084

(0.082-0.093) e (0.082-0.103)

10

0.047

(0.033-0.077)

Median OD

Range

5 8

0.076 0.027

(0.020-0.240) c (0.020-0.084) °

6 4

0.047 0.015

(0.005-0.192) (0.005-0.084)

10

0.016

(0.013-0.034)

n

uc Grade ->2 Grade < 2 CD Grade ->2 Grade < 2 Non-IBD controls Grade 0

ap < 0.04, UC grade ->2 vs. <2; P < 0.03, UC grade ->2 vs. CD grade ->2; P < 0.01, UC grade ->2 vs. non-IBD controls. ~P < 0.02, UC grade < 2 vs. controls with non-IBD controls. cp < 0.04, UC grade ->2 vs. UC grade < 2 ; P < 0.04, UC grade ->2 vs. CD grade >2; P < 0.009, UC grade ->2 vs. non-IBD controls. op < 0.04, UC grade < 2 vs. non-IBD controls and CD grade <2. eNo significant difference, CD grade ->2 vs. grade < 2 and vs. non-IBD controls, both for IgG and IgG1 to TMs.

patients than in both patients with actively inflamed UC (P < 0.009) and with UC in remission (P < 0.04). In CD, IgG and IgG1 antibodies to TMs, produced by LPMCs, did not differ in the presence or absence of active inflammation and also when compared with non-IBD controls (Table 3).

IgG and IgG1 Antibodies Against TMs and Histological Assessment of Mucosal Inflammation in IBD: Comparison With Non-IBD Controls IgG and IgG1 antibodies to TMs produced by LPMCs from the colon of patients with UC in remission (grade 1) or with active histological inflammation (grades 2 and 3) are shown in Figure 3. None of the patients with IBD had grade 4 histological inflammation. LPMCs from all patients with UC, irrespective of their histological grades (grades 1-3), produced higher IgG and IgG1 antibodies to TMs than patients with CD (P < 0.004 to P < 0.05) and non-IBD controls (P < 0.05 to P < 0.002). The median values for IgG antibodies to TMs for grades 1 - 3 were 0.084 (range, 0.067-0.616), 0.213 (range, 0.136-0.754), and 0.188 (range, 0.078-0.302), respectively; and for IgG1 antibodies to TMs the values were 0.020 (range, 0.010-0.084), 0.069 (range, 0.0600.240), and 0.130 (range, 0.020-0.240), respectively. IgG and IgG1 antibodies to TMs produced by LPMCs in patients with UC were higher in actively inflamed mucosa (grades 2 and 3) when compared with the absence of active inflammation (grade 1) (P < 0.02 to P < 0.003). No significant difference was noted between grades 2 and 3 (Figure 3). In patients with CD, the median OD values for IgG antibodies to TMs in mucosa

1.000

,900

IgG

to

TMs

.800

I .700

m

.600

m

_

I

a

.500

m~

Q .400

.

.300

m

O I-

IgG1

to

TMs

2

3

.200 m

.100

.! 1

2

3

1

HISTOLOGIC G R A D E S OF UC COLON INFLAMMATION

Figure 3. Histograms showing the median OD values and range for LPMC production of IgG and IgG1 antibodies to TMs by LPMCs from the colon of patients with UC with histological inflammatory scores of grades 1 (1~), 2 (1@), and 3 (U) (n = 11, 5, and 8, respectively). LPMCs isolated from the colon of patients with UC with histological inflammatory scores of grade 2 and 3 produced higher levels of IgG and IgG1 antibodies to TMs than LPMCs isolated from the colon of patients with UC with inflammatory scores of grade 1. The production of IgG and IgG1 antibodies to TMs did not differ between grades 2 and 3. The results of the statistical analysis by nonparametrical KruskalWallis Test are as follows: IgG to TMs, grade 2 vs. grade 1 (P < 0.02) and grade 3 vs. grade 1 (P < 0.05); IgG1 to TMs, grade 2 vs. grade 1 (P < 0.02) and grade 3 vs. grade 1 (P < 0.003).

10

BIANCONE ET AL.

with active inflammation (grades 2 and 3) and in the absence of active inflammation (grade 1) were similar, 0.101 (range, 0.082-0.108) vs. 0.084 (range, 0.0820.103). Similar results in patients with CD were also observed for IgG1 antibodies to TMs (active vs. inactive inflammation: OD, 0.045; range, 0.005-0.195 vs. OD, 0.010; range, 0.005-0.020). These values did not differ when compared with controls with non-IBD (Table 3), whereas they were lower than in patients with UC with both active inflammation (P < 0.004 for IgG and P < 0.05 for IgG1) and inactive inflammation (P < 0.05 for both IgG and IgG1). The statistical dependence of antibody production (OD) and mucosal inflammatory grades was analyzed in all groups. Because the dependent variable (OD) is continuous and the independent variable (inflammatory score) is ordinal, the statistical relation was not considered in terms of correlation but as a linear trend. A linear trend is expressed by the variance explained by the regression (R2). A positive trend of IgG1 antibodies to TMs production vs. mucosal inflammatory grades was detected in patients with UC, as indicated by the percentage of variance explained by the regression, i.e., R 2 = 55% for the endoscopic score and R 2 = 50% for the histological score.

Discussion In the current study, we have shown that LPMCs and PBMCs in patients with UC produce IgG antibodies against TMs. The production of IgG antibodies to TMs was higher in patients with symptomatic UC when compared with patients with UC in remission, and it was confined mainly to IgG1 subclass. IgG1 is known to be capable of complement activation, and in our study, the mucosal production of IgG1 antibodies to TMs increased in the colon of patients with UC with active inflammation. The background OD values, both by LPMCs and PBMCs, did not differ between patients with CD and non-IBD controls, and it was probably related to nonspecific binding of the supernatants to the tissue extracts. 2° Western blot analysis using supernatants and the enriched TMs was uniformly negative for all groups, including the UC group, possibly because of a very low concentration of IgG antibody in the supernatants derived from the cultured cells of the biopsy specimens and denaturation of the antigenic protein(s) in SDS-PAGE. However, with the sensitive ELISAs, the total IgG and IgG1 antibodies in the supernatants could be measured. Total IgG and IgG1 antibodies were higher in both patients with UC and with CD when compared with non-IBD controls and were higher in the patients with

GASTROENTEROLOGY Vol. 109, No. 1

symptomatic UC than in both patients with UC in remission, confirming previous findings. 16'2° However, the spontaneous production of IgG antibodies to TMs by LPMCs was significantly higher in patients with UC than in patients CD and with non-IBD, and more so in the colon of patients with UC with active inflammation when compared with the colon of patients with UC in remission. It is interesting to note that the production of IgG antibodies to TMs by LPMCs isolated from the rectum and sigmoid colon was significantly higher than from the proximal colon in patients with UC but not in patients with CD and in non-IBD controls. These findings may reflect more severe disease in the rectum and sigmoid colon as is commonly found in patients with UC. This is further supported by our study showing an increased production of both IgG and IgG1 antibodies to TMs in the colon of patients with UC with active inflammation. Such differences were absent in patients with CD and with non-IBD, suggesting disease specificity for UC. Moreover, statistical analysis showed a trend in patients with UC for IgG1 antibodies to TMs vs. the endoscopical and histological degree of mucosal inflammation. Our data also confirm previous studies 2° showing that, in both patients with UC and with CD, the total IgG production by LPMCs is higher in the actively inflamed areas compared with non-IBD patients. However, the LPMC production of IgG and IgG1 antibodies against the enriched TMs did not differ between patients with CD and with non-IBD. Total and TMs-specific IgG production did not increase in PWM-stimulated LPMCs, confirming previous findings21 and suggesting that LPMCs are already maximally stimulated by luminal antigens permeating the mucosa. In the intestinal lesions of patients with IBD, a marked increase in the mucosal IgG immunocytes has been shown and related to the severity of tissue damage. 22-25 The IgG antibody production against various antigens 26-3° has been reported in the colon of patients with UC, but the specific antigen(s) involved in the IgGimmune recognition has not been clarified. The mucosal IgG overproduction could play a pathogenetic role in patients with UC. The mucosal subclass distribution of IgG-producing cells has been shown to be different in patients with UC compared with patients with CD involving the colon. 23'31'32 In CD, the IgG1/IgG2 immunocyte ratio is not significantly different from that in the normal control mucosa, whereas in UC, there is a disproportionate local overproduction of IgG1, both in active and inactive lesions. Indeed, IgG1 antibodies to TMs were significantly higher than non-IBD in patients with UC, even in remission. IgG1 antibodies are more effective

July 1995

in complement activation than IgG2, 33'34 and because of this property, IgG1 antibodies can contribute to the perpetuation of the disease. The deposition of activated complement and igG1 has been shown on the colonic epithelium from patients with active UC. 35 The complement deposition in the colon of patients with CD was not associated with the presence of IgG. 36 Recently, triple color immunofluorescence analysis showed that tissue-bound IgG deposits in the colon of patients with UC colocalized with the P40 and activated complement products, 3v and the degree of activated complement deposition correlated with the degree of inflammation.35 Our study shows that the previously reported UCassociated IgG antibody recognizing the TM-related protein(s) 6'* is actively produced not only by PBMCs but also by LPMCs infiltrating the inflamed UC tissue. This antibody belongs predominantly to IgG1 subclass, and its local production in the mucosa is markedly increased in the actively inflamed colon. These findings suggest that the colonic P40, shown to be a member of the TM family,8 may be involved in the pathogenesis of UC. At least eight different TM isoforms have been identified from human fibroblasts. 9'38 The specific TM isoform(s) present in the human colon epithelium and its full chemical structure are currently unknown. The multiple TM proteins observed in the colonic mucosal extract may be related to various isoforms contributed by the colonic epithelial cells as well as by the nonepithelial stromal tissue and muscularis mucosa. By immunohistochemistry, specific TM isoform(s) have been localized in the small intestinal brush border of chicken in addition to the cell membrane and the cytoplasm of enterocytes.39 Recently, a novel protein, named zipper protein (Z protein) with partial homology to tropomyosin (35%), has been isolated from the brush border of small intestine of the chicken. 4° Further studies focusing on the colonic epithelial TM isoform(s) and TM-related colonic novel protein, such as the Z protein, and their immune responses in UC may provide important biochemical information to explain autoimmunity in patients with UC.

AUTOANTIBODIES FROM MUCOSAL LYMPHOCYTES IN UC

4.

5.

6.

7.

8.

9. 10. 11.

12. 13. 14.

15.

16.

17.

18.

19.

20.

References 1. Broberger O, Perlmann P. In vitro studies of ulcerative colitis. I. Reactions of patients' serum with human fetal colon cells in tissue culture. J Exp Med 1963; 1 1 7 : 7 0 5 - 7 1 5 . 2. Hibi TB, Also M, Ishikawa M. Circulating antibodies to the surface antigens on colon epithelial ceils in ulcerative colitis. Clin Exp Immunol 1983; 5 4 : 1 6 3 - 1 6 8 . 3. Aronson RA, Cooke SL, Roche JK. Sensitization to epithelial antigens in chronic mucosal inflammatory disease. I. Purification, characterization and immunoreactivity of murine epithelial cell-

21.

22.

11

associated components (ECAC). J Immunol 1 9 8 3 ; 1 3 1 : 2 7 9 6 2844. Duerr RH, Targan SR, Landers CJ, LaRusso NF, Lindsay KL, Wiesner RH, Shanahan F. Neutrophil cytoplasmic antibodies: a link between primary sclerosing cholangitis and ulcerative colitis. Gastroenterology 1991; 1 0 0 : 1 3 8 5 - 1 3 9 1 . Das KM, Dubin R, Nagai T. Isolation and characterization of coIonic tissue-bound antibodies from patients with idiopathic ulcerative colitis. Proc Natl Acad Sci USA 1 9 7 8 ; 7 4 : 4 5 2 8 - 4 5 3 2 . Takahashi F, Das KM. Isolation and characterization of a colonic autoantigen specifically recognized by colon tissue-bound IgG from idiopathic ulcerative colitis. J Clin Invest 1 9 8 5 ; 7 6 : 3 1 1 318. Takahashi F, Shah 14, Wise L, Das KM. Circulating antibodies against human colonic extract enriched with a 40 kDa protein in patients with ulcerative colitis. Gut 1990; 3 1 : 1 0 1 6 - 1 0 2 0 . Das KM, Dasgupta A, Mandal A. Autoimmunity to cytoskeletal protein tropomyosin(s): a new clue to the pathogenetic mechanism for ulcerative colitis. J Immunol 1993; 1 5 0 : 2 4 8 7 - 2 4 9 3 . Lees-Miller JP, Helfman DM. The molecular basis for tropomyosin isoform diversity. Bioessays 1 9 9 1 ; 1 3 : 4 2 9 - 4 3 7 . Truelove SC, Witts U. Cortisone in ulcerative colitis. Final report on a therapeutic trial. BMJ 1 9 5 5 ; 2 : 1 0 4 1 - 1 0 4 8 . Prantera C, Davoli M, Lorenzetti R, Pallone F, Marcheggiano A, lannoni C, Marotti S. Clinical and laboratory indicators of extent of ulcerative colitis. J Clin Gastroenterol 1988; 1 0 : 4 1 - 4 5 . Harvey RF, Bradshaw JM. A simple index of Crohn's Disease activity. Lancet 1980;2:514. Pallone F, Ricci R, Boirivant M, Montano S. Measuring the activity of Crohn's Disease. Ital J Gastroenterol 1 9 8 1 ; 1 3 : 5 1 - 5 3 . Gomes P, Du Boulay C, Smith CL, Holdstock G. Relationship between disease activity indices and colonoscopic findings in patients with colonic inflammatory bowel disease. Gut 1986;27:92-95. Bull DM, Bookmann MA. Isolation and functional characterization of human mucosal lymphoid cells. J Clin Invest 1977; 5 9 : 9 6 6 974. Squarcia O, Fais S, Boirivant M, Di Paolo MC, Marcheggiano A, lannoni C, Paoluzi P, Pallone F. Phenotypes and spontaneous immunoglobulin production in mononuclear cells suspensions isolated from colonic biopsies of patients with mild active and quiescent ulcerative colitis. Gastroenterol Clin Biol 1991; 15:194-198. Lin JJC, Hegmann TE, Lin JLC. Differential localization oftropomyosin isoforms in cultured non-muscle cells. J Cell Biol 1988; 1 0 7 : 5 6 3 - 5 7 2 . Cote A, Doucet JP, Trifaro JM. Adrenal medullary tropomyosins: purification and biochemical characterization. J Neurochem 1986;46:1771-1782. Kendall M, Stuart A. Statistical relationship: linear regression and correlation. In: Griffin C, ed. The Advanced Theory of Statistics. Volume 2 . 4 t h ed. London: High Wycombe, 1 9 7 9 : 2 9 8 - 3 3 7 . Ruthlein J, Ibe M, Burghardt W, Mossner J, Auer I0. Immunoglobulin G (IgG), IgG1 and IgG2 determinations from endoscopic biopsy specimens in control, Crohn's Disease and ulcerative colitis subjects. Gut 1992; 3 3 : 5 0 7 - 5 1 2 . MacDermott R, Nash GS, Bertovich M J, Selden MV, Bragdon M J, Beale MG. Alterations of IgM, IgG and IgA synthesis and secretion by peripheral blood and intestinal mononuclear cells from patients with ulcerative colitis and Crohn's disease. Gastroenterology 1 9 8 1 ; 8 1 : 8 4 4 - 8 5 2 . 8randtzaeg P, Baklien K, Bjerke K, Rognum TO, Scott H, Valnes K. Nature and properties of the human gastrointestinal immune system. In: Miller K, Nuklin S, eds. Immunology of the Gastrointestinal Tract. Boca Raton, FL: CRC, 1 9 8 7 : 1 - 8 5 .

12

BIANCONE ET AL.

23. Brandtzaeg P. Immunopathology of Crohn's disease. Ann Gastroenterol Hepatol 1985;21:201-220. 24. Kobayashi K, Asajura H, Hamada Y, Hibi T, Watanabe M, Yoshida T, Watanabe N, Miura S, Also S, Tsuchiya M. T lymphocyte subpopulations and immunoglobulin-containing cells in the colonic mucosa of ulcerative colitis: a morphometric and immunohistochemical study. J Clin Lab Immunol 1988;25:63-68. 25. Baklien K, Brandtzaeg P. Comparative mapping of the local distribution of immunoglobulin-forming cells in ulcerative colitis and Crohn's Disease of the colon. Clin Exp Immunol 1975;22:197209. 26. Elson CO. The immunology of inflammatory bowel disease. In: Kirsner JB, Shorter RG, eds. Inflammatory Bowel Disease. Volume 80. Philadelphia: Lea & Febiger, 1988:97-164. 27. MacDermott RP, Stenson WA. Alterations of the immune system in ulcerative colitis and Crohn's disease. Adv Immunol 1988; 42:285-322. 28. Zinberg J, Vecchi M, Sakamaki S, Das KM. Intestinal tissue associated antigens in the pathogenesis of inflammatory bowel disease. In: Jarnerot G, ed. Inflammatory Bowel Disease. New York: Raven, 1987:67-76. 29. Hibi T, Ohara M, Toda K, Hara A, Ogata H, Iwao Y, Watanabe N, Watanabe M, Hamada Y, Kobayashi K, Also S, Tsuchiya M. In vitro anticolon antibody production by mucosal or peripheral blood lymphocytes from patients with ulcerative colitis. Gut 1990; 31:1371-1376. 30. Das KM, Sakamaki S, Vecchi M. Ulcerative colitis: specific antibodies against a colonic epithelial Mr 40,000 protein. Immunol Invest 1989; 18:459-472. 31. Helgeland L, Tysk C, Jarnerot G, Kett K, Lindberg E, Danielsson D, Andersen SN, Brandtzaeg P. The IgG subclass distribution in rectal mucosa of monozygotic twins with or without inflammatory bowel disease. Gut 1992; 33:1358-1364. 32. Brandtzaeg P, Halstensen T, Kett K. Immunopathology of inflammatory bowel disease. In: MacDermott RP, Stenson WF, eds. Inflammatory Bowel Disease. New York: Elsevier, 1992:95-136.

GASTROENTEROLOGY Vol. 109, No. 1

33. Shakib F, Stanworth DR. Human IgG subclasses in health and disease. Ric Clin Lab 1980;10:561-580. 34. Heiner DC. Significance of immunoglobulin G subclasses. Am J Med 1984; 76:1-6. 35. Halstensen TS, Molines TE, Garred P, Fausa O, Brandtzaeg P. Epithelial deposition of immunoglobulin G1 and activated complement (C3b and terminal complement complex) in ulcerative colitis. Gastroenterology 1990; 98:1264-1271. 36. Halstensen TS, Mollnes TE, Garred P, Brandtzaeg P. Surface epithelium-derived activation and complement differs in Crohn's disease and ulcerative colitis. Gut 1992;33:902-905. 37. Halstensen TS, Das KM, Brandtzaeg P. Epithelial deposits of immunoglobulin G1 and activated complement co-localize with the 40 kDa colonic autoantigen in ulcerative colitis. Gut 1993; 34:650-657. 38. Novy RE, Lin JLC, Lin CS, Lin JJC. Human fibroblast tropomyosin isoforms: characterization of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in normal and transformed cells. Cell Motil Cytoskeleton 1993;25:267-281. 39. Xie L, Hirabayashi T, Miyakazi T. Histological distribution and developmental changes of tropomyosin isoforms in three chicken digestive organs. Cell Tissue Res 1992;269:391-401. 40. Bikle DD, Munson S, Morrison N, Eisman J. Zipper protein, a newly described tropomyosin-iike protein of intestinal brush border. J Biol Chem 1993;268:620-626. Received November 29, 1993. Accepted January 20, 1995. Address requests for reprints to: Kiron M. Das, M.D., Ph.D., University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 1 Robert Wood Johnson Place, New Brunswick, New Jersey 08903. Fax: (908) 235-6156. Supported by research grants NIADDK R01 DK44314 and NIADDK R01 DK47673 from the National Institutes of Health (Bethesda, MD). Presented in part at the annual meeting of the American Gastroenterological Association in May 1991.