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Appearance of killer (K) cells in the mesenteric lymph nodes in Crohn's disease
00165085/78/7502-0218$02.00/O GAETROENTEROWY 75~218-220,1978 Copyright0 1978by the AmericanGastroenterological Association
Vol. 75, No. 2 Printed in U.SA.
APPEARANCE OF KILLER (K) CELLS IN THE MESENTERIC NODES IN CROHN’S DISEASE SVEN
BRITTON,
M.D.,
A.
E.
EKLUND,
M.D.,
AND A.
G.
BIRD,
LYMPH
M.R.C.P.
Department of Immunology and Microbiology, School of Health Sciences, UCLA, Los Angeles, California; Department of Immunobiology, Wallenbergluboratory, Stockholm, gsweden; and Departments of Infectious Diseases and Surgery, Danderyd Hospital, Danderyd, Sweden
The local lymph node in Crohn’s disease contains killer (K) cells which are able to lyse antibody-coated heterologous target cells. K cells are not found in mesenteric lymph nodes from control patients not suffering from Crohn’s disease, whereas they are regularly found in similar amounts in the peripheral blood of both controls and patients with Crohn’s disease. Even mesenteric lymph node cells from areas not macroscopically affected by inflammatory disease show increased K cell activity as compared to control lymph node lymphocytes, suggesting a generalized mesenteric lymph node involvement with regard to K cell activity in Crohn’s disease.
An immunological involvement in the pathology of Crohn’s disease (CD) is suggested by the intense local lymphoglandular enlargement in the regions of intestinal inflammation. However, most studies so far conducted to study immunological parameters in CD patients have used blood lymphocytes as the tool of research. The data obtained utilizing blood lymphocytes are somewhat conflicting, but the consensus is that no gross changes in blood lymphocyte appearance or behavior’ can be seen in these patients as compared to healthy controls or most other chronic inflammatory disease. In a previous article2 findings were reported indicating that the mesenteric lymph node content of thymusderived lymphocytes (T cells) and the proliferative response of these cells to mitogen appeared comparable to lymph node cells from various types of control patients. Now we have studied the capacity of lymph node lymphocytes to kill antibody-coated target cells. The effector lymphocyte in this system is known as a killer (K) cell and does not bear surface markers of B or T lymphocytes.3 We do find that cells with strong ability to kill antibody-coated target cells appear in the local, as well as distant, mesenteric lymph nodes in CD. Such cells were not found in various control lymph nodes. Received May 24, 1977. Accepted January 15, 1978. Address requests for reprints to: Sven Britton, M.D., Department of Immunobiology, Wallenberglaboratory, Lilla Frescati, 104 05 Stockholm 50, Sweden. This study was supported by grants from the Swedish Medical Research Council to Sven Britton and Grant CA 12800 (National Institutes of Health Principal Investigator Professor John Fahey). The authors thank their colleagues at the Department of Surgical Pathology, School of Health Sciences, UCLA, and Department of Surgery, St. Erik’s Hospital, Stockholm, for providing part of the test material.
Materials and Methods Blood and lymph node specimens were obtained during surgery of the patients from the Department of Surgical Pathology, School of Health Sciences, UCLA, Los Angeles, California; Department of Surgery, St. Erik’s Hospital, Stockholm, Sweden; or Department of Surgery, Danderyd Hospital, Danderyd, Sweden. Only cases of clinically and radiologically unequivocal CD were used as cell donors in this study. The diagnosis was in each instance confirmed by histological examination of resected tissue. No borderline cases between CD and ulcerative colitis were entered in the study. None of the patients had been on steroids or cytostatic drugs within 2 months of obtaining the tissue specimen, but most were on sulfasalazine treatment. Mean duration time of the disease was 4.8 years with range 12.6 to 0.6. Cases involving both large and/or small intestine are included. Microscopic granuloma formation was present in resected areas of all CD patients and in most microscopically affected lymph nodes. Control patients were patients with local cancer of the large bowel (7), diverticulitis (3), appendicitis (3), duodenal ulcer (2), cholelithiasis (2), and mesenteric thrombosis (2). Control lymph nodes were macroscopically unaffected by primary disease. Mean age of CD patients was 39.6 * 9.4 and of control patients 84.2 ? 11.0. Surface markers on lymphocytes. Blood lymphocytes were separated from whole blood according to Biiyum.4 Lymph node lymphocytes were prepared as previously described,2 i.e., by gently pressing the node through a sterile stainless steel mesh after taking away the tissue fat surrounding the node. The cells were then collected in Hanks’ solution and separated according to Boyurn so as to treat blood and lymph node lymphocytes identically. Lymph node cells from Crohn’s patients were taken both from grossly affected nodes and from nodes obtained from mesenteric areas not affected by inflammatory disease. Lymph nodes from control patients were only taken from areas that appeared macroscopically normal, i.e., no nodes with evidence of metastatic tumor. The surface identity of lymphocytes from both blood and lymph nodes was determined after separation according to the procedure of Jondal et a1.5 Thymus-derived lymphocytes were 218
August 1978
KILLER
CELLS
IN CROHN’S
allowed to bind sheep erythrocytes (E rosettes) using incubation at 4°C in a pellet overnight and subsequent gentle resuspension before reading. Lymphocytes with surface structures recognizing activated complement (EAC rosettes) were incubated with sheep erythrocytes (SRBC) that had been coated in a two-step procedure with G-200-separated rabbit antisheep IgM antibodies diluted 1:lOOand, thereafter, mouse serum from A/Sri mice (deficient for C5) diluted 1:4 as the complement source. This mixture was incubated 15 to 30 min at 37°C and agitated before reading. Background rosettes formed with SRBC, coated with IgM only, was always ~2% and was subtracted from the given EAC value. Lymphocytes with receptor structures that bind to the Fc portion of IgG (Fc rosettes) were developed by incubating the lymphocytes for 30 min at 37°C with SRBC coated with hyperimmune rabbit antisheep IgG diluted 1:250. After incubation the cells were agitated vigorously and, thereafter, read in a hemocytometer. Cytotoxic assay. Purified lymphocytes as above were exposed to S’Cr-labeled mouse lymphoma cell, as described previously.” In brief, a Gross virus-induced lymphoma of thymic origin from C3H mice was the target cell. It was kept in continuous suspension culture and showed good and reproducible labeling characteristics. For labeling 0.4 &i of sodium chromate (ICN Corporation, Irvine.Calif.) was added to lo7 tumor cells in 1 ml of minimum essential medium supplemented with 10% fetal calf serum. After incubation at 37°C for 90 min the labeled cells were incubated with the effector lymphocytes at a ratio of 1:20 (target-to-effector cell). The tumor cell number was kept constant 104 cells per tube yielding 3000 to 5000 counts per min. Immune rabbit serum (IRS) was obtained by injecting a rabbit four times weekly with 2.5 x lo7 tumor cells and bled 3 days after the last injection. Of the antibody obtained by this procedure, 95% wsa mercaptoethanol-resistant. Rabbit serum or normal control rabbit serum (NRS) was then added to the mixture in O.l-ml volume. The final volume was 0.3 ml. The incubation period was 2.5 hr at 37°C in 5% CO2 in air. The reaction was stopped by adding 1 ml of cold minimum essential medium and the mixture was centrifuged in the cold at 185 x g for 10 min. The supernatant was decanted and the radioactivity therein determined in a y counter (Nuclear-Chicago Corp., Des Plaines, Ill.). Percentage of specific lysis was
TABLE 1. Surface characteristics”
Source of lymphocytes -__.__ CD blood (171
219
DISEASE
calculated according to the formula: % released bv IRS - % released in NRS % specific lysis = % released bv Triton - % released bi NRS (The percentage released by Triton represents the total lysable counts (SO%).) Within the above test period the spontaneous release was 5%, regardless of whether the tumor cells were cultured alone or in the presence of live or killed lymphocytes or heat-inactivated normal or immune rabbit serum.
Results Surface markers in blood and lymph node lymphocytes from Crohn’s patients and controls. A recent extensive study’ has reported no gross difference in proportions or absolute numbers of lymphocytes carrying surface markers when CD patients were compared to controls. Our study confirms this when blood lymphocytes are compared (table 1). The surface marker profile of lymphocytes from mesenteric lymph nodes is different from that found in blood insofar as cells carrying Fc receptors (Fc rosettes) are regularly fewer than those expressing complement receptors (EAC), a ratio opposite to that found in blood (table 1). The purified lymphocytes from regional lymph nodes in CD generally display proportionally more Fc-positive rosettes than do control specimens. Distant lymph nodes, from bowel areas macroscopically not affected by CD, show a profile comparable to controls (table 1). Antibody-dependent cellular cytotoxicity: comparison between lymph node and blood lymphocytes from Crohn’s patients and various controls. Blood lymphocytes from CD patients and controls showed no difference with regard to capacity to lyse antibody-coated target cells (table 2). However, when lymph node lymphocytes were compared, it was clearly shown that those from CD patients killed more efficiently than did control lymph node cells. This was true both when the
of blood and mesenteric lymph node lymphocytes control@
from patients with Crohn’s disease (CD) and various
% of rosette-forming
lymphocytes
E
FC
71.2 ? 3.2
19.0 2 1.6
4 P>
Control blood (19)
70.4 + 2.9
Local CD lymph noded (17)
80.6 k 4.6
Distal CD lymph node’ (6)
79.3 f 8.6
Control lymph node (19)
81.0 t 2.5
t P < 1 P < t P> t
0.1
19.4 c 1.3 0.01 8.9 k 1.4 0.01 3.1kO.8 0.1 2.9 -+ 0.3
EAC d
12.4 r 0.6
P> 0.1 5 13.1 P> 0.1 $ 10.4 P >, 0.1 r 9.7 P> 0.1 t 9.4
-e 0.6 i 0.4 2 0.8 2 0.3
” Rosette-forming ability of lymphocytes against sheep red blood cells (E rosettes), red blood cells coated with IgG antibody (Fc rosettes), and red blood cells coated with IgM plus complement (EAC). Values are mean 2 SE. ’ See Materials and Methods. ’ Purified lymphocytes from indicated origin and tissue. Numbers in parentheses refer to the number of specimens tested. d The regional lymph node. ’ Macroscopically nonaffected lymph nodes.
220
BRITTON
ET AL.
Vol. 75, No. 2
from CD patients as compared to controls. However, we are aware that our data do not tell anything about absolute changes in the lymph node lymphocyte populations. % of specific lysis Source of lymphocyt& Our data indicate, with regard to the regional lymph J 35.4 i 3.1 CD blood (17) nodes in CD, that there is a positive correlation between 0.1 P> the proportion of lymphocytes carrying the Fc receptor Control blood (19) t 34.7 2 3.1 (Fc rosettes) and the cytotoxic potential against anti1 22.7 t 3.8 Local CD lymph node (16) body-coated target cells, a finding similar to that seen PC 0.1 for blood lymphocytes.3 In contrast, the distal mesen$ 14.8 2 4.6 Distal CD lymph node (6) teric lymph nodes (table 2) show increased K cell P < 0.01 activity in CD, although without a significantly inControl lymph node (18) T 3.0 ? 0.6 creased proportion of Fc receptor-bearing cells as com” Calculation according to formula described in Materials and pared to controls. Thus, the correlation of K cell activity Methods section. Values are means + SE. and Fc receptor-bearing cells is not absolute in CD, but b See Materials and Methods. we believe that these findings indicate a generalized ’ Isotope-labeled mouse lymphoma cells. mesenteric lymph node involvement in the disease ’ Hyperimmune rabbit antitarget cell antibody. process as revealed by changes in K cell activity. ” Purified lymphocytes from indicated origin and tissue. Figures in parentheses are numbers of specimens tested. We have treated both patient and control lymph node lymphocytes with trypsin so as to digest any protein (antigen-antibody complex) material that could regional lymph nodes of CD patients and, to a lesser block the receptor and thus shield K cell activity in our extent, the distant macroscopically unaffected CD assay (unpublished data). This is feasible because the lymph node cells were compared to those of control Fc receptor is insensitive to trypsin as revealed by lymph nodes. As K cells did appear in the nodes of CD rerosetting after trypsin treatment (unpublished data). patients, it was clear that the preparation of lymph However, such treatment could not reveal any further node cells as such did not abolish K cell activity. Also, activity in the lymph nodes tested. as the K cell activity in blood from patients and controls The nature of the effector cell in antibody-dependent were similar, it is unlikely that the age differences that cellular cytotoxicity is still obscure. It was initially existed between these two groups were of importance. thought to be bone marrow-derived,3 but recent data9 This is in agreement with our earlier findings6 but now seem to indicate that part of the effector population admittedly we have not completely excluded that nor- could indeed be comprised of activated thymus-derived mal age-dependent changes of K cell activity may lymphocytes carrying the Fc receptor. With the recently appear in lymph nodes in contrast to blood. developed single cell plaque assay for K cell activity,g it should be possible to identify the origin of the lymph Discussion node K cell in CD. In this article we demonstrate that the local as well REFERENCES as distant mesenteric lymph nodes in CD contain effector cells (K) which are capable of lysing antibody-coated 1. Thayer WR, Charland C, Field C: The subpopulations of circulating white blood cells in inflammatory bowel disease. Gastrotarget cells. Such cells are not found in various control enterology 71:379-384, 1976 mesenteric lymph nodes or lymph nodes from other Bird AG, Britton S: No evidence for decreased lymphocyte parts of the human body, the latter finding being reactivity in Crohn’s disease. Gastroenterology 67:926-932, 1974 independently reported by others.7 MacLennan I: Antibody in the induction and inhibition of We think that it is likely that a “blood lymph node lymphocyte cytotoxicity. Transplant Rev 13:67-123, 1972 barrier” exists because the profile of lymphoid cells in Boyum A: Separation of leucocytes from human blood and lymph nodes from various anatomical locations is cerbonemarrow. Stand J Clin Lab Invest Bl(supp1 97): S-109, 1968 tainly different from that found in blood.7 Our data Jondal M, Holm G, Wigzell H: Surface markers on human T and suggest that the K cell is normally “held” by this barrier B lymphocytes. I. A large population of lymphocytes forming but that there is a breakdown in the mesenteric lymph nonimmune rosettes with sheep erythrocytes. J Exp Med 136:207-215, 1972 nodes in CD. A less likely possibility is that K cells mature locally in the lamina propria of disease-affected 6. Elhilali M, Britton S, Brosman S, et al: Critical evaluation of lymphocyte functions in urological cancer patients. Cancer Res bowel. 36:132-137, 1976 At this stage it is premature to speculate about Samarut C, Brochier J, Revillard J: Distribution of cells binding whether this change in cellular composition represents erythrocyte-antibody (EA) complexes in human lymphoid popua link in the tissue-destructive process in CD or merely lations. Stand J Immunol 5221-231, 1976 reflects a collapse of the internal lymph node structure Crohn B, Ginzburg L, Oppenheimer G: Regional enteritis: a secondary to inflammation. The increased cellularity of pathologic and clinical entity. JAMA 99:1323-2819, 1932 mesenteric lymph nodes in CD is, however, lymphoids Wahlin B, Perlmann H, Perlmann P: Analysis by a plaque assay in nature, and we did not find any increase in nonlymof IgG- or IgM-dependent cytotoxic lymphocytes in human blood. J Exp Med 144:1375-1380, 1976 phoid cell elements in the lymph node cell suspensions 2. Percentage of specific lysis” induced by blood or mesenteric lymph node lymphocytes from patients with Crohn’s disease (CD) and various controls” on “K’r-labeled heterologous target cells” in the presence of target cell antibody” TABLE
Vol. 75, No. 2 Printed in U.SA.
APPEARANCE OF KILLER (K) CELLS IN THE MESENTERIC NODES IN CROHN’S DISEASE SVEN
BRITTON,
M.D.,
A.
E.
EKLUND,
M.D.,
AND A.
G.
BIRD,
LYMPH
M.R.C.P.
Department of Immunology and Microbiology, School of Health Sciences, UCLA, Los Angeles, California; Department of Immunobiology, Wallenbergluboratory, Stockholm, gsweden; and Departments of Infectious Diseases and Surgery, Danderyd Hospital, Danderyd, Sweden
The local lymph node in Crohn’s disease contains killer (K) cells which are able to lyse antibody-coated heterologous target cells. K cells are not found in mesenteric lymph nodes from control patients not suffering from Crohn’s disease, whereas they are regularly found in similar amounts in the peripheral blood of both controls and patients with Crohn’s disease. Even mesenteric lymph node cells from areas not macroscopically affected by inflammatory disease show increased K cell activity as compared to control lymph node lymphocytes, suggesting a generalized mesenteric lymph node involvement with regard to K cell activity in Crohn’s disease.
An immunological involvement in the pathology of Crohn’s disease (CD) is suggested by the intense local lymphoglandular enlargement in the regions of intestinal inflammation. However, most studies so far conducted to study immunological parameters in CD patients have used blood lymphocytes as the tool of research. The data obtained utilizing blood lymphocytes are somewhat conflicting, but the consensus is that no gross changes in blood lymphocyte appearance or behavior’ can be seen in these patients as compared to healthy controls or most other chronic inflammatory disease. In a previous article2 findings were reported indicating that the mesenteric lymph node content of thymusderived lymphocytes (T cells) and the proliferative response of these cells to mitogen appeared comparable to lymph node cells from various types of control patients. Now we have studied the capacity of lymph node lymphocytes to kill antibody-coated target cells. The effector lymphocyte in this system is known as a killer (K) cell and does not bear surface markers of B or T lymphocytes.3 We do find that cells with strong ability to kill antibody-coated target cells appear in the local, as well as distant, mesenteric lymph nodes in CD. Such cells were not found in various control lymph nodes. Received May 24, 1977. Accepted January 15, 1978. Address requests for reprints to: Sven Britton, M.D., Department of Immunobiology, Wallenberglaboratory, Lilla Frescati, 104 05 Stockholm 50, Sweden. This study was supported by grants from the Swedish Medical Research Council to Sven Britton and Grant CA 12800 (National Institutes of Health Principal Investigator Professor John Fahey). The authors thank their colleagues at the Department of Surgical Pathology, School of Health Sciences, UCLA, and Department of Surgery, St. Erik’s Hospital, Stockholm, for providing part of the test material.
Materials and Methods Blood and lymph node specimens were obtained during surgery of the patients from the Department of Surgical Pathology, School of Health Sciences, UCLA, Los Angeles, California; Department of Surgery, St. Erik’s Hospital, Stockholm, Sweden; or Department of Surgery, Danderyd Hospital, Danderyd, Sweden. Only cases of clinically and radiologically unequivocal CD were used as cell donors in this study. The diagnosis was in each instance confirmed by histological examination of resected tissue. No borderline cases between CD and ulcerative colitis were entered in the study. None of the patients had been on steroids or cytostatic drugs within 2 months of obtaining the tissue specimen, but most were on sulfasalazine treatment. Mean duration time of the disease was 4.8 years with range 12.6 to 0.6. Cases involving both large and/or small intestine are included. Microscopic granuloma formation was present in resected areas of all CD patients and in most microscopically affected lymph nodes. Control patients were patients with local cancer of the large bowel (7), diverticulitis (3), appendicitis (3), duodenal ulcer (2), cholelithiasis (2), and mesenteric thrombosis (2). Control lymph nodes were macroscopically unaffected by primary disease. Mean age of CD patients was 39.6 * 9.4 and of control patients 84.2 ? 11.0. Surface markers on lymphocytes. Blood lymphocytes were separated from whole blood according to Biiyum.4 Lymph node lymphocytes were prepared as previously described,2 i.e., by gently pressing the node through a sterile stainless steel mesh after taking away the tissue fat surrounding the node. The cells were then collected in Hanks’ solution and separated according to Boyurn so as to treat blood and lymph node lymphocytes identically. Lymph node cells from Crohn’s patients were taken both from grossly affected nodes and from nodes obtained from mesenteric areas not affected by inflammatory disease. Lymph nodes from control patients were only taken from areas that appeared macroscopically normal, i.e., no nodes with evidence of metastatic tumor. The surface identity of lymphocytes from both blood and lymph nodes was determined after separation according to the procedure of Jondal et a1.5 Thymus-derived lymphocytes were 218
August 1978
KILLER
CELLS
IN CROHN’S
allowed to bind sheep erythrocytes (E rosettes) using incubation at 4°C in a pellet overnight and subsequent gentle resuspension before reading. Lymphocytes with surface structures recognizing activated complement (EAC rosettes) were incubated with sheep erythrocytes (SRBC) that had been coated in a two-step procedure with G-200-separated rabbit antisheep IgM antibodies diluted 1:lOOand, thereafter, mouse serum from A/Sri mice (deficient for C5) diluted 1:4 as the complement source. This mixture was incubated 15 to 30 min at 37°C and agitated before reading. Background rosettes formed with SRBC, coated with IgM only, was always ~2% and was subtracted from the given EAC value. Lymphocytes with receptor structures that bind to the Fc portion of IgG (Fc rosettes) were developed by incubating the lymphocytes for 30 min at 37°C with SRBC coated with hyperimmune rabbit antisheep IgG diluted 1:250. After incubation the cells were agitated vigorously and, thereafter, read in a hemocytometer. Cytotoxic assay. Purified lymphocytes as above were exposed to S’Cr-labeled mouse lymphoma cell, as described previously.” In brief, a Gross virus-induced lymphoma of thymic origin from C3H mice was the target cell. It was kept in continuous suspension culture and showed good and reproducible labeling characteristics. For labeling 0.4 &i of sodium chromate (ICN Corporation, Irvine.Calif.) was added to lo7 tumor cells in 1 ml of minimum essential medium supplemented with 10% fetal calf serum. After incubation at 37°C for 90 min the labeled cells were incubated with the effector lymphocytes at a ratio of 1:20 (target-to-effector cell). The tumor cell number was kept constant 104 cells per tube yielding 3000 to 5000 counts per min. Immune rabbit serum (IRS) was obtained by injecting a rabbit four times weekly with 2.5 x lo7 tumor cells and bled 3 days after the last injection. Of the antibody obtained by this procedure, 95% wsa mercaptoethanol-resistant. Rabbit serum or normal control rabbit serum (NRS) was then added to the mixture in O.l-ml volume. The final volume was 0.3 ml. The incubation period was 2.5 hr at 37°C in 5% CO2 in air. The reaction was stopped by adding 1 ml of cold minimum essential medium and the mixture was centrifuged in the cold at 185 x g for 10 min. The supernatant was decanted and the radioactivity therein determined in a y counter (Nuclear-Chicago Corp., Des Plaines, Ill.). Percentage of specific lysis was
TABLE 1. Surface characteristics”
Source of lymphocytes -__.__ CD blood (171
219
DISEASE
calculated according to the formula: % released bv IRS - % released in NRS % specific lysis = % released bv Triton - % released bi NRS (The percentage released by Triton represents the total lysable counts (SO%).) Within the above test period the spontaneous release was 5%, regardless of whether the tumor cells were cultured alone or in the presence of live or killed lymphocytes or heat-inactivated normal or immune rabbit serum.
Results Surface markers in blood and lymph node lymphocytes from Crohn’s patients and controls. A recent extensive study’ has reported no gross difference in proportions or absolute numbers of lymphocytes carrying surface markers when CD patients were compared to controls. Our study confirms this when blood lymphocytes are compared (table 1). The surface marker profile of lymphocytes from mesenteric lymph nodes is different from that found in blood insofar as cells carrying Fc receptors (Fc rosettes) are regularly fewer than those expressing complement receptors (EAC), a ratio opposite to that found in blood (table 1). The purified lymphocytes from regional lymph nodes in CD generally display proportionally more Fc-positive rosettes than do control specimens. Distant lymph nodes, from bowel areas macroscopically not affected by CD, show a profile comparable to controls (table 1). Antibody-dependent cellular cytotoxicity: comparison between lymph node and blood lymphocytes from Crohn’s patients and various controls. Blood lymphocytes from CD patients and controls showed no difference with regard to capacity to lyse antibody-coated target cells (table 2). However, when lymph node lymphocytes were compared, it was clearly shown that those from CD patients killed more efficiently than did control lymph node cells. This was true both when the
of blood and mesenteric lymph node lymphocytes control@
from patients with Crohn’s disease (CD) and various
% of rosette-forming
lymphocytes
E
FC
71.2 ? 3.2
19.0 2 1.6
4 P>
Control blood (19)
70.4 + 2.9
Local CD lymph noded (17)
80.6 k 4.6
Distal CD lymph node’ (6)
79.3 f 8.6
Control lymph node (19)
81.0 t 2.5
t P < 1 P < t P> t
0.1
19.4 c 1.3 0.01 8.9 k 1.4 0.01 3.1kO.8 0.1 2.9 -+ 0.3
EAC d
12.4 r 0.6
P> 0.1 5 13.1 P> 0.1 $ 10.4 P >, 0.1 r 9.7 P> 0.1 t 9.4
-e 0.6 i 0.4 2 0.8 2 0.3
” Rosette-forming ability of lymphocytes against sheep red blood cells (E rosettes), red blood cells coated with IgG antibody (Fc rosettes), and red blood cells coated with IgM plus complement (EAC). Values are mean 2 SE. ’ See Materials and Methods. ’ Purified lymphocytes from indicated origin and tissue. Numbers in parentheses refer to the number of specimens tested. d The regional lymph node. ’ Macroscopically nonaffected lymph nodes.
220
BRITTON
ET AL.
Vol. 75, No. 2
from CD patients as compared to controls. However, we are aware that our data do not tell anything about absolute changes in the lymph node lymphocyte populations. % of specific lysis Source of lymphocyt& Our data indicate, with regard to the regional lymph J 35.4 i 3.1 CD blood (17) nodes in CD, that there is a positive correlation between 0.1 P> the proportion of lymphocytes carrying the Fc receptor Control blood (19) t 34.7 2 3.1 (Fc rosettes) and the cytotoxic potential against anti1 22.7 t 3.8 Local CD lymph node (16) body-coated target cells, a finding similar to that seen PC 0.1 for blood lymphocytes.3 In contrast, the distal mesen$ 14.8 2 4.6 Distal CD lymph node (6) teric lymph nodes (table 2) show increased K cell P < 0.01 activity in CD, although without a significantly inControl lymph node (18) T 3.0 ? 0.6 creased proportion of Fc receptor-bearing cells as com” Calculation according to formula described in Materials and pared to controls. Thus, the correlation of K cell activity Methods section. Values are means + SE. and Fc receptor-bearing cells is not absolute in CD, but b See Materials and Methods. we believe that these findings indicate a generalized ’ Isotope-labeled mouse lymphoma cells. mesenteric lymph node involvement in the disease ’ Hyperimmune rabbit antitarget cell antibody. process as revealed by changes in K cell activity. ” Purified lymphocytes from indicated origin and tissue. Figures in parentheses are numbers of specimens tested. We have treated both patient and control lymph node lymphocytes with trypsin so as to digest any protein (antigen-antibody complex) material that could regional lymph nodes of CD patients and, to a lesser block the receptor and thus shield K cell activity in our extent, the distant macroscopically unaffected CD assay (unpublished data). This is feasible because the lymph node cells were compared to those of control Fc receptor is insensitive to trypsin as revealed by lymph nodes. As K cells did appear in the nodes of CD rerosetting after trypsin treatment (unpublished data). patients, it was clear that the preparation of lymph However, such treatment could not reveal any further node cells as such did not abolish K cell activity. Also, activity in the lymph nodes tested. as the K cell activity in blood from patients and controls The nature of the effector cell in antibody-dependent were similar, it is unlikely that the age differences that cellular cytotoxicity is still obscure. It was initially existed between these two groups were of importance. thought to be bone marrow-derived,3 but recent data9 This is in agreement with our earlier findings6 but now seem to indicate that part of the effector population admittedly we have not completely excluded that nor- could indeed be comprised of activated thymus-derived mal age-dependent changes of K cell activity may lymphocytes carrying the Fc receptor. With the recently appear in lymph nodes in contrast to blood. developed single cell plaque assay for K cell activity,g it should be possible to identify the origin of the lymph Discussion node K cell in CD. In this article we demonstrate that the local as well REFERENCES as distant mesenteric lymph nodes in CD contain effector cells (K) which are capable of lysing antibody-coated 1. Thayer WR, Charland C, Field C: The subpopulations of circulating white blood cells in inflammatory bowel disease. Gastrotarget cells. Such cells are not found in various control enterology 71:379-384, 1976 mesenteric lymph nodes or lymph nodes from other Bird AG, Britton S: No evidence for decreased lymphocyte parts of the human body, the latter finding being reactivity in Crohn’s disease. Gastroenterology 67:926-932, 1974 independently reported by others.7 MacLennan I: Antibody in the induction and inhibition of We think that it is likely that a “blood lymph node lymphocyte cytotoxicity. Transplant Rev 13:67-123, 1972 barrier” exists because the profile of lymphoid cells in Boyum A: Separation of leucocytes from human blood and lymph nodes from various anatomical locations is cerbonemarrow. Stand J Clin Lab Invest Bl(supp1 97): S-109, 1968 tainly different from that found in blood.7 Our data Jondal M, Holm G, Wigzell H: Surface markers on human T and suggest that the K cell is normally “held” by this barrier B lymphocytes. I. A large population of lymphocytes forming but that there is a breakdown in the mesenteric lymph nonimmune rosettes with sheep erythrocytes. J Exp Med 136:207-215, 1972 nodes in CD. A less likely possibility is that K cells mature locally in the lamina propria of disease-affected 6. Elhilali M, Britton S, Brosman S, et al: Critical evaluation of lymphocyte functions in urological cancer patients. Cancer Res bowel. 36:132-137, 1976 At this stage it is premature to speculate about Samarut C, Brochier J, Revillard J: Distribution of cells binding whether this change in cellular composition represents erythrocyte-antibody (EA) complexes in human lymphoid popua link in the tissue-destructive process in CD or merely lations. Stand J Immunol 5221-231, 1976 reflects a collapse of the internal lymph node structure Crohn B, Ginzburg L, Oppenheimer G: Regional enteritis: a secondary to inflammation. The increased cellularity of pathologic and clinical entity. JAMA 99:1323-2819, 1932 mesenteric lymph nodes in CD is, however, lymphoids Wahlin B, Perlmann H, Perlmann P: Analysis by a plaque assay in nature, and we did not find any increase in nonlymof IgG- or IgM-dependent cytotoxic lymphocytes in human blood. J Exp Med 144:1375-1380, 1976 phoid cell elements in the lymph node cell suspensions 2. Percentage of specific lysis” induced by blood or mesenteric lymph node lymphocytes from patients with Crohn’s disease (CD) and various controls” on “K’r-labeled heterologous target cells” in the presence of target cell antibody” TABLE