Digestive Diseases and Sciences, Vol. 36, No. 10 (October 1990), pp. 1453-1458

Two-Color Immunofluorescence and Flow Cytometric Analysis of Lamina Propria Lymphocyte Subsets in Ulcerative Colitis and Crohn's Disease M. SENJU, MD, K.C. WU, MD, Y.R. MAHIDA, MD, and D.P. JEWELL, FRCP

By using two-color immunofluorescence with fluorescein isothiocyanate (FITC) and phycoerythrin (PE) -labelled monoclonal antibodies and multiparameter flow cytometry, we investigated lamina propria lymphocyte subsets of patients with ulcerative colitis (UC) and Crohn's disease (CD). Leu-3/Leu-2 (CD4/CD8) ratio of lamina propria lymphocytes (LPL) of CD (mean +- so: 1.9 +- 0.8, P < 0.01) was significantly decreased compared with controls (3.3 +- 1.1), because o f an increased number of CD8+ lymphocytes. The majority o f lamina propria CD4 + cells were CD4 +, Leu-8- and CD4 +, CD45R- both in controls and IBD tissue. Many lamina propria T lymphocytes were activated, expressing HLA-DR antigen not only in IBD but also in controls. N K cells defined by CDI6 and CD 56 (3.0 +1.4%, P < 0.01) were significantly decreased in patients with UC compared with controls (6.5 +--3.0%). A low proportion of B cells in the intestinal mucosa expressed Leu-8 antigen and CD23 antigen. The proportion of activated B cells of LPL was high in IBD mucosa as well as normal mucosa. These findings suggest that local activation of B cells leads to the loss of the expression o f Leu-8 antigen and CD23. KEY WORDS: ulcerative colitis; Crohn's disease; lymphocytes; flow cytometry.

In the lamina propria of the intestine, T cells, B cells, and cytotoxic effector cells may play an important role in the pathogenesis of UC or CD. The development of monoclonal antibodies to cellsurface antigens has allowed detailed characterization of subpopulations of lymphoid cells. The subsets of lamina propria lymphocytes (LPL), especially the subsets of T cells, have been investigated extensively by immunofluorescence (1-3) and immunoperoxidase techniques (4) on tissue Manuscript received June 20, 1990; revised manuscript received January 23, 1991; accepted January 28, 1991. From the Gastroenterology Unit, Radcliffe Infirmary, Oxford, U.K. Address for reprint requests: Dr. D.P. Jewell, Gastroenterology Unit, Radcliffe Infirmary, Woodstock Road, Oxford, OX2 6HE, U.K.

sections or on isolated lymphoid cells. Flow cytometry utilizes rapid automated counting of large numbers of cells, which permits multiple surface markers to be analyzed. Flow cytometry can also provide quantitative measurement of fluorescence intensity, which can be related to receptor density (5). Hence, monoclonal antibodies and immunofluorescence analysis by flow cytometry provide a highly sensitive and specific approach to the phenotypic analysis of human lymphocyte subsets. The use of simultaneous two-color immunofluorescence that employs two different antibodies labelled with either FITC or PE has added a new dimension to this type of analysis. The aim of this study was to reinvestigate lymphocyte subpopulations in the lamina propria of the

Digestive Diseases and Sciences, Vol. 36, No. 10 (October 1990)

0163-2116/91/1000-1453506.50/09 1991PlenumPublishingCorporation

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SENJU ET AL intestine of patients with U C and CD. T w o - c o l o r i m m u n o f l u o r e s c e n c e staining and multiparameter flow c y t o m e t r y w e r e used.

TABLE 1. SPECIFICITYOF MONOCLONALANTIBODIES Monoclonal antibodies (FITC/PE) HLe-1/Leu-M3

MATERIALS AND METHODS Patient Population. Thirteen patients undergoing colectomy for UC were studied. The group consisted of nine men and four women with a mean age of 42 years (range 17-65 years). All patients with UC were receiving intravenous hydrocortisone at the time of surgery. Four of them had distal disease, which allowed cells to be isolated from the uninflamed portion of the colon as well as from the affected part. Ten patients with CD undergoing intestinal resection (seven inflamed ileum, and six inflamed colon) also were investigated; there were five men and five women, with a mean age of 40 years (range 15-69 years). All patients with CD except one were receiving intravenous hydrocortisone at the time of operation. The diagnosis of UC or CD was based on clinical, endoscopic, histological, and radiological features. As controls, two normal ileal and 13 colonic mucosal samples were obtained from intestine resected for tumor and one for lipoma of the transverse colon. The mucosa was obtained at least 5 cm from the tumor and was macroscopically and histologically normal. This group included 10 men and four women with a mean age of 66 years (range 50-77 years). All the resected specimens were examined histologically and the diagnosis confirmed. Isolation of Lamina Propria Mononuclear Ceils. Lamina propria mononuclear cells were isolated from normal and inflamed mucosa by a modification of the EDTA collagenase technique of Bull and Bookman (6), as previously described (7). Briefly, tissue was obtained from fresh operation resection specimens and transported in cold RPMI 1640 medium (Gibco, Paisley, Scotland). After washing, strips of mucosa were dissected and incubated in 1 mM dithiothreitol solution (DTT; Sigma, Poole, Dorset, U.K.) for 20 min at room temperature. To remove the epithelial cells, mucosal strips were shaken with 5 mM ethylene-diaminetetraacetic acid (EDTA; BDH, Poole, Dorset, U.K.) at 37~ for half an hour on three occasions with washes with calcium- and magnesium-free Hanks' balanced salt solution (CMF-HBSS, Flow Laboratories, Irvine, Scotland) in between. After the final wash with HBSS with calcium and magnesium, the mucosa was minced into 1-mm pieces and digested in RPMI 1640 containing collagenase (from Clostridium histolyticum, Boehringer, Mannheim GmbH, Germany) at a concentration of 1 mg/ml as well as 10% fetal calf serum, 100 units/ml of penicillin, 100 l~g/ml of streptomycin, and 50 vg/ml of gentamicin at 37~ for 3 hr. The digested tissue was passed through a nylon mesh (Henry Simon, Stockport, Cheshire, U.K.) with a pore size of 100 ~m in diameter to obtain the cells. After thorough washing, mononuclear cells were obtained by centfifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). The yield (median and range) of mononuclear cells was 10.6 x 106 (2.2-17.6 x 106) for control specimens and 22.5 x 106 (7.7-36.0 x 106) for inflammatory bowel disease. Viability

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IgGJIgG2

Antigen cluster designation CD45/CD14 -/-

Leu-3/Leu-2 Leu-3/Leu-8

CD4/CD8 CD4/-

Leu-18/Leu-3 Leu-2/Leu-8 Leu-4/HLADR Leu-4/IL-2R Leu-7/Leu-2 Leu-7/Leu-4 Leu-4/Leu11+19 Leu-16/Leu-1 Leu-16/Leu-8

CD45R/CD4 CD8/CD3/-

Leu-16/Leu-20 Leu-21/Leu-12

CD3/CD25 CD57/CD8 CD57/CD3 CD3/CD16, CD56 CD20/CD5 CD20/CD20/CD23 CDw78/CD19

Target subset For electronic gate setting for lymphocytes For negative control and nonspecific binding CD4/CD8 ratio Suppressor-inducer and helper-inducer T cells Suppressor-inducer T cells CD8+ T subsets Activated T cells Activated T cells CD8 subsets CD3 subsets NK cells and CTL Leu-l+ B cells Leu-8+ and Leu-8- B cells FceR+ B cells Activated B cells

was greater than 85% as determined by trypan blue exclusion. Monodonal Antibodies. Fifteen pairs of monoclonal antibodies conjugated with FITC and PE were used. All monoclonal antibodies were prepared at the Becton Dickinson Monoclonal Center, Inc. (Mountain View, California). The specificities of these reagents used in this study are presented in Table 1. They were all directly conjugated with FITC or PE. Two-Color Direct Immunofiuorescence Staining of LPL. The suspension of LPL was adjusted to 2 x 107 cells/ml. Then 20 irl of each monoclonal reagent was placed into a 12 x 75-mm polystyrene tube (Falcon Plastics, Oxnard, California) and 50 ~1 of the cell suspension (1 x 106 cells) was added to each tube. The tubes were incubated for 30 min on ice in the dark. The samples were washed once in cold Dulbeccos' modified phosphate buffered saline (PBS) without calcium and magnesium (Gibco) containing 0.1% sodium azide. The cell sediment was resuspended in 0.5% paraformaldehyde in PBS. The fixed cells were stored at 4~ in the dark until analysis. Two-Color Flow Cytometry. Analysis by two-color flow cytometry was performed with a FACScan (Becton Dickinson, FACS Division, Sunnyvale, California). A Consort 30 computer system (Becton Dickinson, FACS Division) was used for the flow cytometric data acquisition and analysis. Twenty thousand cells were acquired in list mode for each sample for two-color analysis. The optimum LPL gate for an individual patient was set using a LeucoGATE (Table 1), and subsequent samples for the same patient were analyzed using this gate. A Simultest Control reagent (IgG1 FITC + IgG2 PE, Becton Dickinson) was used to set the optimum fluorescence quadrant markers for each patient and to monitor nonspecific staining. Percentages were calculated based on the number of lymphocytes found in each quadrant. Interassay Digestive Diseases and Sciences, Vol. 36, No. 10 (October 1990)

LYMPHOCYTE SUBSETS IN IBD TABLE 2. PHENOTYPE OF LAMINA PROPRIA LYMPHOCYTES DEFINED BY MONOCLONAL ANTIBODIES

Total lymphocytes (%, mean • SD) Monoclonal antibodies CD8 CD4 CD3 CD20 CD4/CD8

Ulcerative colitis

Controls 18,2 54.2 68.1 13.2 3.3

+• • -----

6.0 8.0 11.3 8.4 1.1

17.1 51.6 71.6 15.7 3.6

--- 6.6 • 9.2 • 6.7 --- 6.1 --- 2.0

TABLE 3. PROPORTION OF T-CELL SUBSETS AND T CELLS BEARING ACTIVATION MARKERS IN LAMINA PROPRIA AS A PERCENTAGE OF TOTAL LYMPHOCYTES (MEAN -----SD)

Controls

Crohn' s disease 30.1 49.4 76.2 10.6 1.9

------• •

12.4" 6.4 6.0"~ 5.7 0.8:~

*P < 0.01. t P < 0.05. * P < 0.001.

CD8+, CD8+, CD4+, CD4+, CD4+, CD4+, CD3+, CD3+,

Leu-8+ Leu-8Leu-8+ Leu-8CD45R+ CD45RHLADR+ IL-2R+

1.1 12.3 10.4 42.7 5.5 49.2 38.4 11.6

----------+• •

2.3 5.8 9.3 9.7 7.0 8.5 14.3 6.1

Ulcerative colitis 1.4 13.6 16.3 32.8 9.1 45.4 34.3 11.9

• • -----------

1.2 5.9 9.1 8.8t 6.6 10.3 9.5 4.0

Crohn's disease 1.3 21.6 7.1 36.2 3.7 43.8 34.5 10.8

• --------• • ---

0.8 12.8" 4.5 11.0 3.6 10.6 10.4 4.3

*CD v s control P < 0.05. tTUC v s control p < 0.05.

reproducibility was checked using CaliBRITE beads (Becton Dickinson) and AutoCOMP software (Becton Dickinson). Statistical Methods. Statistical analysis of results was performed by Using Student's paired or unpaired t test, following a one~way analysis of variance, and a probability less than 5% was considered to be significant. RESULTS

Single-Color Fluorescence. Table 2 shows the percentages (mean +- SD) of singly fluorescent LPL reacting with monoclonal antibodies, Leu-2 (CD8), Leu-3 (CD4), Leu-4 (CDD), Leu-16 (CD20), as well as the CD4/CD8 ratio in normal mucosa and in mucosa from patients with UC or CD. There was no significant difference in the percentage of CD4+ lymphocytes between normal mucosa and IBD mucosa. In contrast, the percentage of CD8+ lymphocytes was significantly higher in CD (P < 0.01) than in controls. Consequently, the CD4/CD8 ratio was significantly decreased in patients w!th CD (P < 0:001) compared with that in the controFpopulation. Two-Color Fluorescence. The phenotype of CDS+ cells and CD4+ cells was investigated in more detail using two-color immunofluorescent staining. The percentage of CD8+, Leu-8- Cells was higher in patients with CD than in controls (P < 0.05) (Table 3). The proportion of CD8+, Leu-8+ lymphocytes remained unchanged, suggesting that the main cause of the low CD4/CD8 ratio in CD was an increase in the proportion of CD8+, Leu-8- cells. Although the proportion of CD4+, Leu-8+ cells (suppressor,inducer T) iia UC was increased compared with controls, this difference was not statistically significant (Table 3). The proportion of CD4+, Leu-8- cells (helper-inducer T) was decreased in UC compared with controls (P < 0.05). Similarly, using a panel of CD4, Leu-18 (CD45R) the percentage of CD4+, CD45R+ lymphocytes in Digestive Diseases and Sciences. Vol. 36, No. I0 (October 1990)

patients with UC was increased and that of CD4+, CD45R- cells was decreased compared with controis, but these differences were not statistically significant. Leu-8+ cells and CD45R+ cells among T lymphocytes were scarce in the lamina propria, and the majority of CD4+ T cells in the intestine were L e u - 8 - CD45R-. There were no significant differences in the proportions of CD3+, HLA-DR+ cells and CD3+, IL-2R+ cells present in the intestinal mucosa between controls and IBD. Many lamina propria T cells in normal mucosa (38.4 +- 14.3%), uninflamed portion of UC colon (34.5 --- 5.8%), and inflamed mucosa (UC: 34.3 --- 9.5%, CD: 34~5 +-- 10.4%) expressed HLA-DR antigens (Table 3). However, the percentage of IL-2R+ activated T cells in the uninflamed portion of UC colon (22.3 --- 9.5%) was significantly increased compared with controls (P < 0.05). NK cells (CD3-, CD16+, CD56+) were significantly decreased in inflamed mucosa of UC colon (P < 0.01) but not in the uninflamed portion (5.3 --0.6%) (Table 4). There was no significant difference in the proportion of cytotoxic T lymphocytes (CD3+, CD16+, CD56+) between controls and patients with IBD. CD57+, CD8+ cells and CD57, CD3 + cells were very few in the intestine, and the proportion of these cells remained unchanged in controls and IBD. Although the examination of B-cell subsets showed no change in the proportion of Leu-l+ (CD5) B cells, the majority of B cells in the intestinal mucosa did not possess the CD5 antigen on the cell surface (Table 5). Lamina propria B lymphocytes from both inflamed and normal mucosa rarely expressed Leu-8 or FceR (CD23) antigens, and there were no differences between the groups.

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SENJU ET AL TABLE 4. N K CELLS AND CYTOTOXIC T LYMPHOCYTES IN LAMINA PROPRIA

Total lymphocytes (%, mean • so)

Controls Ulcerative colitis C r o h n ' s disease

CD3-, Leu11+19+ (NK cells)

CD3+, Leu-11+19+ (CTL)

Leu-7+, CDS+

Leu-7+, CD3+

6.5 • 3.0 3.0 • 1.4' 4.6 -+ 2.7

5.2 -+ 2.4 4.0 --- 2.1 4.8 -+ 2.5

2.6 • 1.2 4.0 • 3.6 2.4 • 1.8

3.9 • 1.5 5.0 • 1.8 4.2 • 3.4

*P < 0.01.

Leu-21 (CDw78) recognizes an antigen expressed by activated B cells (8). There was no significant difference in the percentage of Leu-21+ B cells in controls and IBD, but the ratio of activated to total B lymphocytes (Leu-12 + (CD19) B cells) in CD (0.42 --+ 0.14) and in the uninflamed portion of UC colon (0.56 -+ 0.33) was significantly increased compared with controls (P < 0.05). Although the ratio in the inflamed mucosa of UC was increased, the difference was not statistically significant. DISCUSSION Analysis of intestinal mucosal lymphocytes presents many difficulties. Immunofluorescent or immunoperoxidase techniques on tissue sections are limited in terms of quantitation of absolute numbers or proportions of individual subsets. Furthermore, if a panel of antibodies to a wide range of surface markers is to be used, the cell populations have to be analyzed over a series of adjacent sections, which is unsatisfactory if inflammation is patchy. Analysis of isolated populations of lamina propria lymphocytes overcomes these problems, but there is no satisfactory way of determining subtle changes in subpopulations of cells resulting from the isolation procedure itself. Despite these difficulties, studies on isolated cell populations have given similar results to those based on tissue sections with respect to the major T-cell subsets and activation markers (3). By using a recently developed system for simultaneous two-color immunofluorescence staining and multiparameter flow cytometry (FACScan), we

have investigated LPL subsets in patients with UC and CD. These studies have employed a wide range of monoclonal antibodies in combination to identify lymphocyte subsets in the intestinal mucosa. Several previous studies have shown that the CD4/CD8 ratio of lamina propria in IBD remains unchanged (3, 4, 9). In the present study, CD4+ lymphocytes in IBD showed no change, but there was an increase in CD8+ cells so that the ratio of CD4/CD8 of patients with CD, but not UC, was significantly decreased. The finding is in keeping with the results of James et al (12), who found a higher proportion of Leu2a+ (CD8+) cells in the lamina propria of patients with Crohn's disease compared with controls, although the CD4/CD8 ratio was not given. Within the CD8 + lymphocytes, there was an increased proportion of CD8+, Leu-8- cells in inflamed mucosa of CD. Since CD8+, Leu-8+ lymphocytes from peripheral blood collaborate with CD8+, Leu-8- cells to generate suppression of B-cell differentiation (10, 11), more detailed studies of the CD8+ subpopulation of isolated LPL are required. Within the CD4+ T cell population, CD4+ cells may be divided into two subpopulations by Leu-8 and CD45R. In patients with CD, there was no substantial difference in the phenotypes of CD4+ cells in lamina propria defined by Leu-8 and CD45R compared with controls, which is in agreement with a previous report (12). In contrast, in inflamed mucosa of UC, CD4+, Leu-8+ cells (suppressorinducer T) were increased, and CD4+, Leu-8cells (helper-inducer T) were decreased. From pre-

TABLE 5. B-LYMPHOCYTE SUBSETS IN LAMINA PROPRIA

Total lymphocytes (%, mean +- SD)

Controls Ulcerative colitis C r o h n ' s disease

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CD20+, CD5+ (Leu-l+ B)

CD20+, Leu-8+ (Leu-8+ B)

CD20+, CD23+ (Fc~_R+ B)

CD19+, Leu-21 + (activated B)

1.1 + 0.7 1.1 --- 0.4 1.2 • 0.9

2.3 -+ 3.6 2.8 -+ 3.9 1.2 -+ 1.2

1.1 -+ 1.6 1.7 -+ 2.1 0.7 --- 1.2

4.5 -+ 4.1 5.4 -+ 3.1 4.0 -+ 2.7

Digestive Diseases and Sciences, Vol. 36, No. 10 (October 1990)

L Y M P H O C Y T E S U B S E T S IN IBD

vious studies, the major function of CD4+, Leu-8+ phenotype from peripheral blood is to act as suppressor-inducer T cells, which activate suppressorprecursor cells to become suppressor-effector cells (10, 13). In contrast, cells with a CD4+, Leu-8phenotype from peripheral blood have a helper function and are involved in B-cell differentiation into plaque-forming cells (10, 11). Recently it has been shown that the respective functions of CD4+ L e u - 8 - cells and CD4+ Leu-8+ cells isolated from lamina propria are similar to their functions in peripheral blood (14). The CD45R+ antigen defines a similar subset of CD4+ cells as Leu-8 (15). Results obtained using the CD4/CD45R combination confirmed the increase in suppressor-inducer cells and the decrease in helper-inducer cells shown by the CD4/Leu-8 studies. These changes in CD4+ and CD8+ T-lymphocyte subpopulations in the lamina propria of UC patients suggest that the immune status in the intestinal mucosa may be shifted towards helper function for immunoglobulin production (10, 16). There was no significant difference in the proportion of activated T cells defined by HLA-DR or IL-2R (CD25) between normal mucosa and inflamed mucosa. However, compared with peripheral blood (17-19), LPL from both inflamed mucosa and normal mucosa contained a larger proportion of activated T cells bearing HLA-DR or CD25. The explanation of these findings is unknown, but it is possible that this pattern of T cell activation in lamina propria is a result of exposure to antigens or mitogens in the intestinal environment. This is supported by a previous report that the expression of early activation antigens defined by 4F2 and T9 was high in lamina propria lymphocytes in inflamed mucosa of CD as well as in normal noninflamed mucosa (19). The paucity or absence of NK cells in intestinal mucosa using CD57 and CD16 monoclonal antibodies has confirmed previous studies (20-22). Recent studies, however, have demonstrated that the cytotoxic effector cells in lamina propria express Leu-19 (23, 24), which is present on both CD3 + and C D 3 cells and has been associated with NK and other forms of non-MHC-restricted cytotoxicity (22). In the present study, it was not possible to analyze CD16 and CD56 separately. However, only 5.2% of normal LPL were CD3+, L e u - l l + , Leu-19+ and 6.5% were C D 3 - , L e u - l l + , Leu-19+ (non-MHCrestricted NK cells (22). Similar proportions were found for LPL from patients with CD but signifiDigestive Diseases and Sciences, Vol. 36, No. 10 (October 1990)

cantly lower proportions of NK cells were found for UC patients. The CD5 antigen is expressed on 50% of peripheral blood lymphocytes, and this proportion does not change in patients with UC or CD (23). The function of this antigen is not clear but may define a cell with poor capability of differentiating into an antibody-producing cell (24). Therefore, it is of interest that few CD5+ cells were found in the lamina propria of inflamed or noninflamed mucosa. Activation of B cells is thought to be associated with a loss of Leu-8 expression (25). Thus, the absence of Leu-8+ B cells in lamina propria, even in uninflamed mucosa, suggests that the mucosal B cells are already activated. This is confirmed by the increased proportion of B cells expressing Leu-21 (CDw78), another marker of B-cell activation (26, 27), which was particularly marked in inflamed mucosa. Thus, LPL differ markedly in phenotype from peripheral blood cells. LPL show a paucity of suppressor-inducer T cells (CD4+, Leu-8+ and CD4+, CD45R+), and both T and B cells in lamina propria bear activation markers. LPL from patients with CD show an increased proportion of CD8+, Leu-8- lymphocytes whereas LPL from UC show a reduction in CD4+, Leu-8- cells. Whether these changes have significance for local immunoregulatory control can only be determined by functional assays since cell function cannot be assumed from its phenotype. ACKNOWLEDGMENTS We are grateful to Dr. Frank Hulstaert and Dr. James Lowder (Becton Dickinson, Immunocytometry Systems Europe, Erembodegem, Belgium) for helpful discussion and for the supply of antibodies. We are also grateful to Mr. N. Mortensen and Mr. M. Kettlewell, our surgeons, who provided the resection specimens.

REFERENCES 1. Selby WS, Janossy G, Goldstein G, Jewell DP: T lymphocyte subsets in human intestinal mucosa: the distribution and relationship to MHC-derived antigens. Clin Exp Immunol 44:453-458, 1981 2. Allison MC, Poulter LW, Dhillon AP, Pounder RE: Immunohistological studies of surface antigen on colonic lymphoid cells in normal and inflamed mucosa. Gastroenterology 99:421-430, 1990 3. Selby WS, Janossy G, Bofill M, Jewell DP: Intestinal lymphocyte subpopulations in inflammatory bowel disease: An analysis by immunohistological and cell isolation techniques. Gut 25:32-40, 1984

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S E N J U ET A L 4. Hirata I, Berrebi G, Austin LL, Keren D, Dobbins WO III: Immunohistologieal characterization of intraepithellal and lamina propria lymphocytes in control ileum and colon and in inflammatory bowel disease. Dig Dis Sci 31:593-603, 1986 5. Parks DR, Lanier LL, Herzenberg LA: Flow cytometry and fluorescence activated cell sorting (FACS). In Hand Book of Experimental Immunology, Vol 1. DM Weir (ed). Oxford, Blackweil Scientific Publications, 1986, pp 29.1-29.21 6. Bull DM, Bookman MA: Isolation and functional characterization of human intestinal mueosal lymphoid cells. J Clin Invest 59:966-974, 1977 7. Gibson PR, Hermanowicz A, Verhaar HJJ, Ferguson DJP, Bernal AL, Jewr DP: Isolation of intestinal mononuclear ceils: Factors released which affect lymphocyte viability and function. Gut 26:60-68, 1985 8. Kikutani H, Kimura R, Nakamura H, Sato R, Muraguchi A, Kawamura N, Hardy RR, Kishimoto T: Expression and function of an early activation marker restricted to human B cells. J Immunol 136:4019,-4026, 1986 9. James SP, Fiocchi C, Graeff AS, Strober W: Immunoregulatory function of lamina propria T cells in Crohn's disease. Gastroenterology 88:1143-1150, 1985 10. Gatenby PA, Kansas GS, Chen YX, Evans RL, Engleman EG: Dissection of !mmunoregulatory subpopulations of T lymphocytes within the helper and suppressor sublineages in man. J Immunol 129:1997-2000, 1982 11. Kansas GS, Wood GS, Fishwild DM, Engleman EG: Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-Leu-8. J Immunol 134: 2995-3002, 1985 12. James SP, Fiocchi C, Graeff AS, Strober W: Phenotypic analysis of lamina propfia lymphocytes: Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn's disease and control patients. Gastroenterology 91:1483-1489, 1986 13. Damle NK, Mohagheghpour N, Engleman EG: 'Soluble antigen-primed inducer T ceils ~ictivate antigen-specific suppressor T cells in the absence of antigen-pulsed accessory Cells: Phenotypic definition of suppress0r-inducer and suppressor-effector cells~.J Immunol 132:644-650, 1984 14. KarlDf ME, Strober W, Fiocchi C, Zeitz M, James SP: CD4 positive Leu-8 negative helper inducer T cells predominate in the human intestinal, lamina propria. J Immunol 141:30293036, 1988 15. Morimoto C, Letvin NL, Distaso JA, Aldrich WR, Schlossman SF: The isolation and characterization of the human suppressor inducer T ,cell subset. J Immunol 134:1508-1515, 1985 16. MacDermott RP, Nash GS, Bertovich MJ, Seiden MV, Bragdon MJ, Beale MG: Alterations of IgM, IgG and IgA

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17.

18.

19.

20. 21.

22.

23.

24.

25.

26.

27.

synthesis and secretion by peripheral blood and intestinal mononuclear cells from patients with ulcerative colitis and Crohn's disease. Gastroenterology 81:844-852, 1981 Uchiyama T, Broder S, Waldmann TA: A monoclonal antibody (anti-Tat) reactive with activated and functionally mature human T cells. 1. Production of anti-Tac monoclonal antibody and distribution of Tae (+) ceils. J Immunol i26:1393-1397, 1981 Zeitz M, Greene WC, Peffer NJ, James SP: Lymphocytes isolated from the intestinal lamina propria of normal nonhuman primates have increased expression of genes associated with T-cell activation. Gastroenterology 94:647-655, 1988 Pallone F, Fais S, Squarcia O, Biancone L, Pozzilli P, Boirivant M: Activation of peripheral blood and intestinal lamina propria lymphocytes in Crohn's disease. In vivo state of activation and in vitro response to stimulation as defined by the expression of early activation antigens. Gut 28:745753, 1987 Shanahan F, Brogan M, Targan S: Human mueosal cytotoxic effector cells. Gastroenterology 92:!951-1957, 1987 Shanahan F, Deem R, Nayersina R, Leman B, Targan S: Human muc0sal T-cell cytotoxicity. Gastroenterology 94:960-967, 1988 Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH: The relationship of CD16 (Leu-I 1) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol 136:4480-4486, 1986 Senju M, Jewell DP: Two 'colour immunofluorescence and flow cytometric analysis of peripheral blood lymphocyte subsets in ulcerative colitisand Crohn's disease. Gut (in pres s) Sidman CL, Shultz LD, Hardy RR, Hayakawa K, Herzenberg LA: Production of immunoglobulin isotypes by Ly-1 + B cells in viable motheaten and n.ormal mice. Science 232:1423-1425, 1986 Kansas GS, Wood GS, Engleman EG: Maturational and functional diversity of human B lymphocytes delineated with anti-Leu-8. J Immunol 134:3003-3006, !985 ' Kikutani H, Kimura R, Nakamura H, Sato R, Muraguchi A, Kawamura N, Hardy RR, Kishimoto T; Expression and fuacti0n of an early activation marker restricted to human B cells. J Immunol 136:4019-4026, 1986 Kikutani H, Nakamura H, Sato R, Kimura T, Yamasaki K, Hardy RR, Kishimoto T: Delineation and characterization of human B cell subpopulations at various stages of activation by using a B celLspecific monoclonal antibody. J Immunol 136:4027-4034, 1986

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