Peptides, Vol. 11, pp. 879-884. © Pergamon Press plc, 1990. Printed in the U.S.A.
0196-9781/90 $3.00 + .00
Gastrointestinal Neuropeptides Suppress Human Colonic Lamina Propria Lymphocyte DNA Synthesis Y O R A M E L I T S U R *l A N D G O R D O N D. L U K
Departments of Medicine and *Pediatrics, Wayne State University, Children's Hospital of Michigan and Harper Hospital, Detroit, MI 48201 R e c e i v e d 12 April 1990
ELITSUR, Y. AND G. D. LUK. Gastrointestinalneuropeptides suppress human colonic laminapropria lymphocyte DNA synthesis. PEPTIDES 11(5) 879-884, 1990.--Gastrointestinal neuropeptides have been shown to modulate the circulatory immune system, but their effect on the mucosal immune system is not well defined. We studied the effect of VIP, SOM, S-P and Bomb on thymidine incorporation into human colonic lamina propria lymphocyte (LPL) DNA. Physiologic concentrations of VIP, SOM, S-P and Bomb significantly suppressed thymidine incorporation into Con A-stimulated human LPL. These neuropeptides did not affect DNA synthesis when LPL were induced with phorbol ester (PDB) and calcium ionophore (ionomycin). Our data suggest that a) VIP, SOM, S-P, and Bomb may have a regulatory role in the human mucosal immune system, and b) Bomb should be added to the list of neuropeptides which affect the gut immune system. Mucosal immune system LPL Neuropeptides Somatostatin (SOM) Bombesin (Bomb)
Vasoactive intestinal polypeptide (VIP)
THE gastrointestinal neuropeptides have been implicated in the functions of the gastrointestinal tract and its associated immune system (26). It has been postulated that the gastrointestinal neuropeptides, such as somatostatin (SOM), vasoactive intestinal polypeptide (VIP) and substance P (S-P) are secreted by terminal peripheral neurons in the intestinal tract and modulate the local immune system (23). Indeed, several investigators have shown that VIP, S-P, and SOM are capable of altering DNA synthesis and immunoglobulin production of peripheral lymphocytes in human and animal models (19). In other studies, bombesin (Bomb) had a mild suppressive or stimulatory effect on thymidine incorporation into Con A-induced mouse lymphocytes and a significant inhibition on IL-2-induced CTLL-2 cell proliferation (4,10). In addition, specific receptors for these hormones have been reported on human and murine peripheral lymphocytes, as well as on a human lymphoblast cell line (IM-9) (23). However, the effect of these gastrointestinal neuropeptides on the human gut immune system has not been described. We studied the effect of VIP, SOM, S-P, and Bomb on the proliferation of human colonic lamina propria lymphocytes (LPL), as quantitated by DNA synthesis. Our results show that all four gastrointestinal neuropeptides studied significantly suppressed DNA synthesis in Con A-induced LPL at very low concentrations (10 - 9 to 10-13 M). Gastrointestinal neuropeptides did not affect DNA synthesis in LPL which were stimulated with the combination of phorbol ester (PDB) and
Substance P (S-P)
calcium ionophore (ionomycin). To our knowledge, this is the first report showing that physiologic concentrations of VIP, SOM, S-P and bombesin can affect human mucosal immune function. METHOD
Materials Tissue culture media and supplies were obtained from Gibco Inc., Grand Island, NY. Collagenase, VIP, SOM, S-P, bombesin, dithiothreitol (DTT), phorbol 12,13 dibutyrate (PDB), trypan blue, ethidium bromide, acridine orange, and other chemicals were purchased from Sigma Chemical Co., St. Louis, MO. Calcium, ionophore (ionomycin) was purchased from Calbiochem, La Jolla, CA. Con A was purchased from ICN ImmunoBiologicals, Lisle, IL. 3H-Thymidine (specific activity 85 ~Ci/ mmole) was purchased from NEN, Dupont, Boston, MA. Fluorescein-conjugated goat anti-mouse IgG, Leu 4 (CD3), Leu ~2 (CD19) were purchased from Becton Dickinson, Mountain View, CA. OKT4 (CD4) and OKT8 (CD8) were purchased from Coulter Immunology, Hialeah, FL.
Human Colon Specimens Fifteen human colon specimens were obtained from adult patients who underwent colon resection. The patients ranged in
XRequests for reprints should be addressed to Dr. Elitsur, Wayne State University School of Medicine, 2136 Scott Hall, 540 E. Canfield, Detroit, MI 48202.
879
880
age from 36 to 82 (mean age, 68). Only histologically normal tissue at least 5 cm from the diseased area was used for the study. In addition, a flow cytometry characterization of LPL component was done on the cells obtained from each specimen to assure normal mucosal immune cell population. The study was approved by the Human Investigation Committee of Wayne State University.
Lamina Propria Lymphocyte Isolation Technique Isolation of LPL was done according to Bull and Bookman with modification and was previously described elsewhere (1,3). LPL isolation was begun within 30 minutes of surgical resection. Briefly, the mucosa was stripped from its musculature and was cut into small pieces (0.5 x 0 . 5 cm). To remove mucus, tissue was treated for 10 minutes with calcium-magnesium-free Hanks balanced salt solution (CMF-HBSS) containing 0.1 M dithiothreitol (DTT). Mucosa was then incubated in ethylenediaminetetraacetate (EDTA) for 90 minutes to remove epithelial cells. After washing twice with CMF-HBSS, the tissue fragments were digested overnight with media (RPMI-1640) containing 10% fetal calf serum, antibiotics (penicillin 10,000 U/ml, amphotericin-B 25 ~xg/ml, streptomycin 10 mg/ml) and collagenase (0.1 mg/100 ml). The cell suspension was filtered and the filtrate was washed three times and placed over a Ficoll-Hypaque gradient to isolate the LPL. The lymphocytes were then recovered, washed and resuspended in RPMI-1640 containing 10% human AB serum and 1% (v/v) antibiotics (standard cell culture solution) for further analysis. Cell viability was determined by trypan blue exclusion test or ethidium bromide and acridine orange staining.
Flow Cytometry Characterization of LPL The normal mucosal immune cell population in each colon specimen was assessed by flow cytometry. Characterization of T helper/T suppressor cell ratio of the LPL was then determined. The flow cytometry was performed according to previously described methods (9). Briefly, 0.05 ml of LPL suspension (5-10 million cells) was incubated with 0.02 ml of Leu 4 (CD3) and Leu 12 (CD19) (B&D) and 0.005 ml of T4 (CD4) and T8 (CD8) (Coulter) at 40C for 30 min. After washing, the cells were incubated with 1:25 dilution of fluorescein-conjugated goat anti-mouse IgG (B&D) at 4°C for another 30 minutes. After washing again, the cells were analyzed on a Becton-Dickinson FACS Analyzer, using a 540 nm filter. The percentage of cells reactive for each antigen was recorded. This was performed in triplicate for all 15 LPL specimens.
Neuropeptide Studies LPL were induced in standard cell culture media with Con A at a final concentration of 5 Ixg/ml. The induced LPL were then placed in 96-well fiat-bottom multititer plates at a cell density of 2 x 105 cells/well. The LPL were then incubated in the presence or absence of graded concentrations (range 1 0 - 1 5 - 1 0 - 7 M) of the tested gastrointestinal neuropeptides (VIP, SOM, S-P, Bomb). Control cultures were treated similarly, except that gastrointestinal neuropeptides were not present. Each experiment was done in triplicate and repeated 6-7 times. All LPL cultures in the multititer plates were then incubated at 37°C, in 5% CO,, to complete 5 days of incubation with the tested neuropeptide (the 5-days incubation resulted in significant suppression by neuropeptides, as determined by the time course studies described below). Six hours prior to their harvest, 3 H thymidine (0.5 ixCi/well) was added to the cultures. Thymidine
ELITSUR AND LUK
incorporation assay was then carried out by standard methods as previously described from our laboratory (3). Briefly, the cells were collected over a glass paper filter (Mash-2) and washed and DNA precipitated by TCA. Radioactivity was then quantitated with a Packard 1500 liquid scintillation beta-counter. As previously described (3, 9, 22), results are expressed as the ratio of CPM incorporated into the test cultures compared to its control according to the following formula: Fraction of control = CPM(test-background)/CPM(control-background). The time course experiments were performed as described above, except that the Con A-stimulated LPL were incubated with the tested neuropeptides, and harvested after 2, 3, 4, and 5 days of incubation for thymidine incorporation assay. The time course experiments were done in triplicate and repeated 3 times. To further investigate the intracellular mechanisms by which VIP, SOM, S-P and Bomb might affect thymidine incorporation into LPL, other intracellular mitogens (PDB + ionomycin) were used. In this set of experiments LPL (2.5 x 10 6 cells/culture tube) were stimulated with PDB (10 8 M) and ionomycin (0.5 IxM) and were incubated (5% CO 2, 37°C) for 1,2, 3, and 5 days. Cells were washed with standard culture medium to remove the mitogens, and were resuspended in standard culture medium containing V1P, SOM, S-P or Bomb (10- ~ M). Cells were then transferred into the standard multititer plate (2 × 105 cells/well) and the experiments continued as described above for standard neuropeptide studies. The time course study was done in triplicate. The neuropeptide inhibition of PDB and ionomycin-induced LPL was performed three separate times, each time in triplicate.
Determination of ¢H-Thymidine Ent~ Into LPL To exclude the possibility that the inhibition of 3H-thymidine incorporation is due to alteration of 3H-thymidine entry into the cells, a 3H-thymidine uptake time course study was carried out as previously described by Schell et al. (22). Briefly, Con Astimulated LPL (2.5 × 10 6 cell/ml) were incubated with VIP, SOM, S-P and Bomb (conc. 10- ~~ M) in 5 ml culture tubes for 5 days. 3H-Thymidine (2.5 ixCi/tube) was added, and the cells were harvested at 1, 3 and 6 hours after thymidine pulsing. At each time point cells were spun and washed twice with PBS, and the DNA was precipitated with 5% ice-cold trichloroacetic acid (TCA). The tubes were then spun and the supernatant (soluble cell fraction) and the pellet (insoluble cell fraction) were collected. The pellet was filtered through fiberglass filter paper and was washed with excess amounts of ice-cold 5% TCA to remove nonspecific radioactive thymidine binding. Radioactivity of both cell fractions was quantitated in liquid scintillation fluid. This study was performed in triplicate. The experimental conditions for this set of studies are markedly different from the LPL DNA synthesis studies described above. Direct comparison of results with those studies cannot be made.
Statistical Analysis Analysis of variance (ANOVA) followed by multiple comparisons test was used to assess the differences between groups, p Values less than 0.05 were considered significant. RESULTS
Lamina Propria Lymphocyte Characterization Cell viability, as assessed by trypan blue exclusion and by ethidium bromide/acridine orange staining, was greater than 90% in each LPL preparation. The T cell subpopulations were then assessed in all 15 colon specimens used in this study. T helper
GI NEUROPEPTIDES INHIBIT H U M A N LPL DNA SYNTHESIS
881
1.2]
TABLE 1 FLOW CYTOMETRYCHARACTERIZATIONOF LPL (%) No. Mean SEM
~
CD19
CD3
CD4
CD8
CD4/CD8
Viability
10.6 1.9
64.0 3.2
40.4 3.3
19.8 2.0
2.52 0.4
93.1 0.9
Flow cytometry study on LPL: Flow cytometry study was done on LPL preparation by cell surface antigen labeling using flow cytometry as described in the Method section. Characterizations were performed on all 15 colon specimens. Data expressed as percentage (mean +_ SEM). CD19--pan B cells. CD3--pan T cells. CD4-T helper/inducer cells. CD8--T suppressor/cytotoxic cells.
(CD4) cells were the major T cell component in the LPL and the mean CD4/CD8 ratio was 2.52:1 (Table 1). This is similar to the results obtained from other normal LPL specimens as previously described (3).
Effect of Gastrointestinal Neuropeptides on Thymidine Incorporation Incubation of unstimulated LPL with VIP, SOM, S-P, or Bomb did not alter thymidine incorporation into LPL DNA (data not shown). On the other hand, VIP, SOM, S-P, and Bomb (conc. 10- 13-10 7 M) significantly suppressed thymidine incorporation into LPL DNA [F(4,20)=130.2, 67.8, 118.8, 73.6 for VIP, SOM, S-P, and Bomb, respectively, p
0196-9781/90 $3.00 + .00
Gastrointestinal Neuropeptides Suppress Human Colonic Lamina Propria Lymphocyte DNA Synthesis Y O R A M E L I T S U R *l A N D G O R D O N D. L U K
Departments of Medicine and *Pediatrics, Wayne State University, Children's Hospital of Michigan and Harper Hospital, Detroit, MI 48201 R e c e i v e d 12 April 1990
ELITSUR, Y. AND G. D. LUK. Gastrointestinalneuropeptides suppress human colonic laminapropria lymphocyte DNA synthesis. PEPTIDES 11(5) 879-884, 1990.--Gastrointestinal neuropeptides have been shown to modulate the circulatory immune system, but their effect on the mucosal immune system is not well defined. We studied the effect of VIP, SOM, S-P and Bomb on thymidine incorporation into human colonic lamina propria lymphocyte (LPL) DNA. Physiologic concentrations of VIP, SOM, S-P and Bomb significantly suppressed thymidine incorporation into Con A-stimulated human LPL. These neuropeptides did not affect DNA synthesis when LPL were induced with phorbol ester (PDB) and calcium ionophore (ionomycin). Our data suggest that a) VIP, SOM, S-P, and Bomb may have a regulatory role in the human mucosal immune system, and b) Bomb should be added to the list of neuropeptides which affect the gut immune system. Mucosal immune system LPL Neuropeptides Somatostatin (SOM) Bombesin (Bomb)
Vasoactive intestinal polypeptide (VIP)
THE gastrointestinal neuropeptides have been implicated in the functions of the gastrointestinal tract and its associated immune system (26). It has been postulated that the gastrointestinal neuropeptides, such as somatostatin (SOM), vasoactive intestinal polypeptide (VIP) and substance P (S-P) are secreted by terminal peripheral neurons in the intestinal tract and modulate the local immune system (23). Indeed, several investigators have shown that VIP, S-P, and SOM are capable of altering DNA synthesis and immunoglobulin production of peripheral lymphocytes in human and animal models (19). In other studies, bombesin (Bomb) had a mild suppressive or stimulatory effect on thymidine incorporation into Con A-induced mouse lymphocytes and a significant inhibition on IL-2-induced CTLL-2 cell proliferation (4,10). In addition, specific receptors for these hormones have been reported on human and murine peripheral lymphocytes, as well as on a human lymphoblast cell line (IM-9) (23). However, the effect of these gastrointestinal neuropeptides on the human gut immune system has not been described. We studied the effect of VIP, SOM, S-P, and Bomb on the proliferation of human colonic lamina propria lymphocytes (LPL), as quantitated by DNA synthesis. Our results show that all four gastrointestinal neuropeptides studied significantly suppressed DNA synthesis in Con A-induced LPL at very low concentrations (10 - 9 to 10-13 M). Gastrointestinal neuropeptides did not affect DNA synthesis in LPL which were stimulated with the combination of phorbol ester (PDB) and
Substance P (S-P)
calcium ionophore (ionomycin). To our knowledge, this is the first report showing that physiologic concentrations of VIP, SOM, S-P and bombesin can affect human mucosal immune function. METHOD
Materials Tissue culture media and supplies were obtained from Gibco Inc., Grand Island, NY. Collagenase, VIP, SOM, S-P, bombesin, dithiothreitol (DTT), phorbol 12,13 dibutyrate (PDB), trypan blue, ethidium bromide, acridine orange, and other chemicals were purchased from Sigma Chemical Co., St. Louis, MO. Calcium, ionophore (ionomycin) was purchased from Calbiochem, La Jolla, CA. Con A was purchased from ICN ImmunoBiologicals, Lisle, IL. 3H-Thymidine (specific activity 85 ~Ci/ mmole) was purchased from NEN, Dupont, Boston, MA. Fluorescein-conjugated goat anti-mouse IgG, Leu 4 (CD3), Leu ~2 (CD19) were purchased from Becton Dickinson, Mountain View, CA. OKT4 (CD4) and OKT8 (CD8) were purchased from Coulter Immunology, Hialeah, FL.
Human Colon Specimens Fifteen human colon specimens were obtained from adult patients who underwent colon resection. The patients ranged in
XRequests for reprints should be addressed to Dr. Elitsur, Wayne State University School of Medicine, 2136 Scott Hall, 540 E. Canfield, Detroit, MI 48202.
879
880
age from 36 to 82 (mean age, 68). Only histologically normal tissue at least 5 cm from the diseased area was used for the study. In addition, a flow cytometry characterization of LPL component was done on the cells obtained from each specimen to assure normal mucosal immune cell population. The study was approved by the Human Investigation Committee of Wayne State University.
Lamina Propria Lymphocyte Isolation Technique Isolation of LPL was done according to Bull and Bookman with modification and was previously described elsewhere (1,3). LPL isolation was begun within 30 minutes of surgical resection. Briefly, the mucosa was stripped from its musculature and was cut into small pieces (0.5 x 0 . 5 cm). To remove mucus, tissue was treated for 10 minutes with calcium-magnesium-free Hanks balanced salt solution (CMF-HBSS) containing 0.1 M dithiothreitol (DTT). Mucosa was then incubated in ethylenediaminetetraacetate (EDTA) for 90 minutes to remove epithelial cells. After washing twice with CMF-HBSS, the tissue fragments were digested overnight with media (RPMI-1640) containing 10% fetal calf serum, antibiotics (penicillin 10,000 U/ml, amphotericin-B 25 ~xg/ml, streptomycin 10 mg/ml) and collagenase (0.1 mg/100 ml). The cell suspension was filtered and the filtrate was washed three times and placed over a Ficoll-Hypaque gradient to isolate the LPL. The lymphocytes were then recovered, washed and resuspended in RPMI-1640 containing 10% human AB serum and 1% (v/v) antibiotics (standard cell culture solution) for further analysis. Cell viability was determined by trypan blue exclusion test or ethidium bromide and acridine orange staining.
Flow Cytometry Characterization of LPL The normal mucosal immune cell population in each colon specimen was assessed by flow cytometry. Characterization of T helper/T suppressor cell ratio of the LPL was then determined. The flow cytometry was performed according to previously described methods (9). Briefly, 0.05 ml of LPL suspension (5-10 million cells) was incubated with 0.02 ml of Leu 4 (CD3) and Leu 12 (CD19) (B&D) and 0.005 ml of T4 (CD4) and T8 (CD8) (Coulter) at 40C for 30 min. After washing, the cells were incubated with 1:25 dilution of fluorescein-conjugated goat anti-mouse IgG (B&D) at 4°C for another 30 minutes. After washing again, the cells were analyzed on a Becton-Dickinson FACS Analyzer, using a 540 nm filter. The percentage of cells reactive for each antigen was recorded. This was performed in triplicate for all 15 LPL specimens.
Neuropeptide Studies LPL were induced in standard cell culture media with Con A at a final concentration of 5 Ixg/ml. The induced LPL were then placed in 96-well fiat-bottom multititer plates at a cell density of 2 x 105 cells/well. The LPL were then incubated in the presence or absence of graded concentrations (range 1 0 - 1 5 - 1 0 - 7 M) of the tested gastrointestinal neuropeptides (VIP, SOM, S-P, Bomb). Control cultures were treated similarly, except that gastrointestinal neuropeptides were not present. Each experiment was done in triplicate and repeated 6-7 times. All LPL cultures in the multititer plates were then incubated at 37°C, in 5% CO,, to complete 5 days of incubation with the tested neuropeptide (the 5-days incubation resulted in significant suppression by neuropeptides, as determined by the time course studies described below). Six hours prior to their harvest, 3 H thymidine (0.5 ixCi/well) was added to the cultures. Thymidine
ELITSUR AND LUK
incorporation assay was then carried out by standard methods as previously described from our laboratory (3). Briefly, the cells were collected over a glass paper filter (Mash-2) and washed and DNA precipitated by TCA. Radioactivity was then quantitated with a Packard 1500 liquid scintillation beta-counter. As previously described (3, 9, 22), results are expressed as the ratio of CPM incorporated into the test cultures compared to its control according to the following formula: Fraction of control = CPM(test-background)/CPM(control-background). The time course experiments were performed as described above, except that the Con A-stimulated LPL were incubated with the tested neuropeptides, and harvested after 2, 3, 4, and 5 days of incubation for thymidine incorporation assay. The time course experiments were done in triplicate and repeated 3 times. To further investigate the intracellular mechanisms by which VIP, SOM, S-P and Bomb might affect thymidine incorporation into LPL, other intracellular mitogens (PDB + ionomycin) were used. In this set of experiments LPL (2.5 x 10 6 cells/culture tube) were stimulated with PDB (10 8 M) and ionomycin (0.5 IxM) and were incubated (5% CO 2, 37°C) for 1,2, 3, and 5 days. Cells were washed with standard culture medium to remove the mitogens, and were resuspended in standard culture medium containing V1P, SOM, S-P or Bomb (10- ~ M). Cells were then transferred into the standard multititer plate (2 × 105 cells/well) and the experiments continued as described above for standard neuropeptide studies. The time course study was done in triplicate. The neuropeptide inhibition of PDB and ionomycin-induced LPL was performed three separate times, each time in triplicate.
Determination of ¢H-Thymidine Ent~ Into LPL To exclude the possibility that the inhibition of 3H-thymidine incorporation is due to alteration of 3H-thymidine entry into the cells, a 3H-thymidine uptake time course study was carried out as previously described by Schell et al. (22). Briefly, Con Astimulated LPL (2.5 × 10 6 cell/ml) were incubated with VIP, SOM, S-P and Bomb (conc. 10- ~~ M) in 5 ml culture tubes for 5 days. 3H-Thymidine (2.5 ixCi/tube) was added, and the cells were harvested at 1, 3 and 6 hours after thymidine pulsing. At each time point cells were spun and washed twice with PBS, and the DNA was precipitated with 5% ice-cold trichloroacetic acid (TCA). The tubes were then spun and the supernatant (soluble cell fraction) and the pellet (insoluble cell fraction) were collected. The pellet was filtered through fiberglass filter paper and was washed with excess amounts of ice-cold 5% TCA to remove nonspecific radioactive thymidine binding. Radioactivity of both cell fractions was quantitated in liquid scintillation fluid. This study was performed in triplicate. The experimental conditions for this set of studies are markedly different from the LPL DNA synthesis studies described above. Direct comparison of results with those studies cannot be made.
Statistical Analysis Analysis of variance (ANOVA) followed by multiple comparisons test was used to assess the differences between groups, p Values less than 0.05 were considered significant. RESULTS
Lamina Propria Lymphocyte Characterization Cell viability, as assessed by trypan blue exclusion and by ethidium bromide/acridine orange staining, was greater than 90% in each LPL preparation. The T cell subpopulations were then assessed in all 15 colon specimens used in this study. T helper
GI NEUROPEPTIDES INHIBIT H U M A N LPL DNA SYNTHESIS
881
1.2]
TABLE 1 FLOW CYTOMETRYCHARACTERIZATIONOF LPL (%) No. Mean SEM
~
CD19
CD3
CD4
CD8
CD4/CD8
Viability
10.6 1.9
64.0 3.2
40.4 3.3
19.8 2.0
2.52 0.4
93.1 0.9
Flow cytometry study on LPL: Flow cytometry study was done on LPL preparation by cell surface antigen labeling using flow cytometry as described in the Method section. Characterizations were performed on all 15 colon specimens. Data expressed as percentage (mean +_ SEM). CD19--pan B cells. CD3--pan T cells. CD4-T helper/inducer cells. CD8--T suppressor/cytotoxic cells.
(CD4) cells were the major T cell component in the LPL and the mean CD4/CD8 ratio was 2.52:1 (Table 1). This is similar to the results obtained from other normal LPL specimens as previously described (3).
Effect of Gastrointestinal Neuropeptides on Thymidine Incorporation Incubation of unstimulated LPL with VIP, SOM, S-P, or Bomb did not alter thymidine incorporation into LPL DNA (data not shown). On the other hand, VIP, SOM, S-P, and Bomb (conc. 10- 13-10 7 M) significantly suppressed thymidine incorporation into LPL DNA [F(4,20)=130.2, 67.8, 118.8, 73.6 for VIP, SOM, S-P, and Bomb, respectively, p
