Abstract
We have developed an enzymatic technique for isolating human intestinal mucosal lymphoid cells. This method was found to be superior to mechanical methods in regard to cell yield and survival. It is based on treating mucosa with serum-free solutions containing collagenase and deoxyribonuclease, followed by isolating the lymphoid cells through centrifugation steps involving fetal calf serum and ficoll-hypaque. Exposure of peripheral blood lymphocytes to the components of the enzymatic solution did not appreciably alter their uptake of tritiated thymidine in the presence or absence of mitogens. Application of the method to derive lymphoid cells from Crohn's disease, ulcerative colitis, and normal intestinal mucosa has shown that gut mucosal lymphocytes from inflammatory bowel disease (1) exceed the number of those from normal mucosa by a factor of 3 to 5; (2) show different degrees of tritiated thymidine uptake, spontaneously and in response to mitogens, depending upon the time they are harvested during the dissociation process; (3) are better stimulators than responders in the allogeneic mixed lymphocyte reaction; (4) generate suppressor cell activity comparable to that of peripheral blood lymphocytes; (5) cannot, in contrast to peripheral blood lymphocytes, generate antibody-dependent cell mediated cytotoxicity; and (6) produce an average of 5 times more IgM than equal numbers of peripheral blood lymphocytes.
Similar content being viewed by others
References
Deodhar SD, Michener WM, Farmer RG: A study of the immunologic aspects of chronic ulcerative colitis and transmural colitis. Am J Clin Pathol 51:591–597, 1969
Perlman P, Broberger O:In vitro studies of ulcerative colitis. II. Cytotoxic action of white blood cells from patients on human fetal colon cells. J Exp Med 117:717–733, 1963
Shorter RG, Cardoza M, Huizenga KA, ReMine SG, Spencer RJ: Further studies ofin vitro cytotoxicity of lymphocytes for colonic epithelial cells. Gastroenterology 57:30–35, 1969
Sachar DB, Taub RN, Ramachandar K, Meyers S, Forman SP, Douglas SD, Janowitz HD: T and B lymphocytes and cutaneous anergy in inflammatory bowel disease. Ann NY Acad Sci 278:565–573, 1976
Strickland RG, Korsmeyer S, Soltis RI, Wilson ID, Williams RC: Peripheral blood T and B cells in chronic inflammatory bowel disease. Gastroenterology 67:569–577, 1974
Strickland RG, Friedler EM, Henderson CA, Wilson ID, Williams RC: Serum lymphocytotoxins in inflammatory bowel disease. Clin Exp Immunol 21:384–393, 1975
Hodgson HJF, Potter BJ, Jewell DP: Immune complexes in ulcerative colitis and Crohn's disease. Clin Exp Immunol 29:187–196, 1977
Chasing the cause of Crohn's disease (editorial). Br Med J 1:929, 1977
Meuwissen SGM, Geltkamp-Wroom TM, De LaRiviere AB, Von Dem Borne AEG, Kr, Tytgat GN: Analysis of the lymphoplasmacytic infiltrate in Crohn's disease with special reference to identification of lymphocyte subpopulations. Gut 17:770–780, 1976
Baklien K, Brandtzaeg P: Comparative mapping of the local distribution of immunoglobulin-containing cells in ulcerative colitis and Crohn's disease of the colon. Clin Exp Immunol 22:197–220, 1975
Clancy R: Isolation and kinetic characterization of mucosal lymphocytes in Crohn's disease. Gastroenterology 70:177–180, 1976
Shortman K, Williams N, Adams P: The separation of different cell classes from lymphoid organs. V. Simple procedures for removal of cell debris, damaged cells and erythroid cells from lymphoid cell suspension. J Immunol Methods 1:273–287, 1973
Li CY, Yam LT, Lam KW: Acid phosphatase isoenzyme in human leukocytes in normal and pathologic conditions. J Histochem Cytochem 18:473–481, 1970
Shou L, Schwartz SA, Good RA: Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors. J Exp Med 143:1100–1110, 1976
Eckhardt R, Kloos P, Dierich MP, Meyer Zum Buschenfelde KH: K-lymphocytes (killer-cells) in Crohn's disease and acute viral hepatitis. Gut 18:1010–1016, 1977
Perlman P, Perlman H: Contactual lysis of antibody-coated chicken erythrocytes by purified lymphocytes. Cell Immunol 1:300–315, 1970
Pollack SB: Effector cells for antibody-dependent cell mediated cytotoxicity. I. Increased cytotoxicity after priming with BCG-SS. Cell Immunol 29:373–381, 1977
Sundeen JT, Krakauer RS: Low level IgM quantitative assay by solid phase immunofluorescence. J Immunol Methods 26:229–244, 1979
Seifter S, Harper E: Collagenases. Methods in Enzymology. GE Perlman, L Lorand (eds). New York, Academic Press, 1970, pp 613–635
Knowles DM, Hoffman T, Ferrarini M, Kunkel HG: The demonstration of acid α-naphthyl acetate esterase activity in human lymphocytes: Usefulness as a T-cell marker. Cell Immunol 35:112–123, 1978
Johnson GJ, Russell PA: Reaction of human lymphocytes in culture to components of the medium. Nature 208:343–345, 1965
Rudzik O, Bienenstock J: Isolation and characterization of gut mucosal lymphocytes. Lab Invest 30:260–266, 1974
Mavligit GM, Jubert AV, Gutterman JU, McBride CM, Hersh EH: Immune reactivity of lymphoid tissue adjacent to carcinoma of the ascending colon. Surg Gynecol Obstet 139:409–412, 1974
Goodacre RL, Bienenstock J, Singal DP, Davidson RA: Morphological and functional characteristics of human intestinal lymphoid cells isolated by a mechanical technique. Gastroenterology 76:300–308, 1979
Bull DM, Bookman MA: Isolation and functional characterization of human intestinal mucosal lymphoid cells. J Clin Invest 59:966–974, 1977
Pretlow TG, II, Weir EE, Zettergren JG: Problems connected with the separation of different kinds of cells. Int Rev Exp Pathol 14:91–204, 1975
Russell SW, Doe WF, Hoskins RG, Cochrane CG: Inflammatory cells in solid murine neoplasma. I. Tumor disaggregation and identification of constitutent inflammatory cells. Int J Cancer 18:322–330, 1976
Willson JKV, Jr, Zaremba JL, Pitts A, Pretlow TG: A characterization of human tonsillar lymphocytes after separation from other tonsillar cells in an isokinetic gradient of ficoll in tissue culture medium. Am J Pathol 83:341–355, 1976
Pumper RW, Fagan P, Taylor WG: Trypsin sensitivity of mammalian cells grown in serum-free medium. In Vitro 6:266–268, 1971
Vischer TL: Stimulation of mouse B lymphocytes by trypsin. J Immunol 113:48–62, 1974
Jondal M, Holm G, Wigzell H: Surface markers of human T and B lymphocytes. I. A large population of lymphocytes forming non-immune rosettes with sheep red blood cells. J Exp Med 136:207–215, 1972
Preud'homme JL, Seligman M: Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40:777–794, 1972
Tan T: The effect of collagenase on surface markers of human peripheral lymphocytes. Br J Dermatol 95:361–364, 1976
Madden RE, Burke D: Production of viable single cell suspensions from solid tumors. J Natl Cancer Inst 27:841–855, 1961
Bernardi G, Ehrlich SD, Thiery TP: The specificity of deoxyribonucleases and their use in nucleotide sequence studies. Nature (New Biol) 246:36–40, 1973
von Boxel JA, Paget SA: Predominantly T cell infiltrate in rheumatoid synovial membranes. N Engl J Med 293:517–520, 1975
Price PA: The essential role of calcium in the activity of bovine pancreatic deoxyribonuclease. J Biol Chem 205:1981–1986, 1975
Shortman K, Williams N, Jackson H, Russell P, Byrt P, Diener, E: The separation of different cell classes from lymphoid organs. IV. The separation of lymphocytes from phagocytes on glass bead columns and its effect on subpopulations of lymphocytes and antibody forming cells. J Cell Biol 48:566–579, 1971
Shortman K: Separation methods for lymphocyte subpopulations. Cont Topics Mol Immunol 3:161–203, 1974
Kohn RR, Rollerson E: Aging of human collagen in relation to susceptibility to the action of collagenase. J Gerontol 15:10–14, 1960
Singal DP, O'Neill M, Clancy R, Bienenstock J: Functional T cells in rabbit gut mucosal lymphocytes. Gut 17:325–327, 1976
Siegal FP, Siegal M: Inhibition of plasma cell formation by leucocytes from hypogammaglobulinemic patients. Clin Res 23:297A, 1975
Abdou NI, Sagawa A, Pascual E, Herbert J, Sadghee S: Suppressor T cell abnormality in idiopathic systemic lupus erythematosus. Clin Immunol Immunopathol 6:192–199, 1976
Twomey JJ, Laughter AJ, Farrow S, Douglass CG: Hodgkin's disease. An immunodepleting and immunosuppressor disorder. J Clin Invest 56:467–475, 1975
Stobo JD, Paul S, Van Scoy RE, Hermans PE: Suppressor thymus derived lymphocytes in fungal infection. J Clin Invest 57:319–328, 1976
Hodgson HJF, Wands JR, Isselbacher KJ: Decreased suppressor cell activity in inflammatory bowel disease. Clin Exp Immunol 32:451–458, 1978
Stobo JD, Tomasi TB, Huizenga KA, Spencer RJ, Shorter RG:In vitro studies of inflammatory bowel disease. Surface receptors of the mononuclear cells required to lyse allogeneic colonic epithelial cells. Gastroenterology 70:171–176, 1976
Clancy R, Pucci A: Absence of K cells in human gut mucosa. Gut 19:273–276, 1978
Jensen KB, Golterman N, Jarnum S, Weeke B, Westergaard H: IgM turnover in Crohn's disease. Gut 11:223–228, 1970
Author information
Authors and Affiliations
Additional information
This work was supported by a grant (CRP 535) from the Committee for Clinical Research Projects of the Cleveland Clinic Foundation.
Rights and permissions
About this article
Cite this article
Fiocchi, C., Battisto, J.R. & Farmer, R.G. Gut mucosal lymphocytes in inflammatory bowel disease. Digest Dis Sci 24, 705–717 (1979). https://doi.org/10.1007/BF01314469
Issue Date:
DOI: https://doi.org/10.1007/BF01314469