- Email: [email protected]
Increased central memory T cells in patients with chronic pancreatitis
Original Paper Received: September 8, 2003 Accepted after revision: July 2, 2004 Published online: April 21, 2005
Pancreatology 2005;5:177–182 DOI: 10.1159/000085269
Increased Central Memory T Cells in Patients with Chronic Pancreatitis Måns Grundstena Guang-Zhi Liub Johan Permerta Peter Hjelmstromb Jon A. Tsaia a Center for Surgical Sciences, Department of Surgery, and b Department of Neurotec, Division of Neurology, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden
Key Words Chronic pancreatitis Central memory T cells CCR7 CD45RA Pancreatic resection
flammatory site. We suggest that the increase of central memory T lymphocytes may be important for maintaining the inflammatory process in chronic pancreatitis. Copyright © 2005 S. Karger AG, Basel and IAP
Abstract Background/Aims: A dysregulated immune response has been suggested to be important for the pathogenesis of chronic pancreatitis (CP). Formation of immunological memory is based on the differentiation of naïve T lymphocytes to memory T lymphocytes after exposure to antigens and specific cytokines. The aim of this study was to analyze peripheral blood mononuclear cells (PBMCs) in patients with CP for different T lymphocyte subsets including naïve and memory T cells. Methods: PBMCs from 9 patients who had undergone pancreatic resection due to CP, 9 CP patients who had not been resected and 9 healthy controls were analyzed by flow cytometry. Results: Patients with CP had a skewed distribution of T lymphocytes, with an increased level of CCR7+/CD45RA– central memory T lymphocytes compared to healthy controls. Nonresected CP patients and subjects who had undergone pancreatic resection due to CP had similar levels of central memory T lymphocytes. Conclusion: Our results indicate that the dysregulation of the immune system in chronic pancreatitis seems to persist even after removal of large parts of the local in-
© 2005 S. Karger AG, Basel and IAP 1424–3903/05/0053–0177$22.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
Introduction
Chronic pancreatitis is a disease charaterized by painful progressive destruction of the pancreatic gland with loss of its exocrine and endocrine functions [1]. The pathophysiological mechanisms are not fully understood, although components of the immune system have been implicated as key elements in the disease [2]. The pancreas is infiltrated by lymphocytes in chronic pancreatitis [3–6] and this appears to at least partially be mediated by upregulation of chemokines in the inflamed gland [7]. In the peripheral blood of chronic pancreatitis patients, several studies have shown a dysregulated distribution of lymphocytes that in some cases is reversed after pancreatic resection [8–11]. T lymphocytes appear to play a key role in the inflammatory process in chronic pancreatitis and constitute a large part of both the cell population infiltrating the pancreas and the dysregulated cell population in peripheral blood. Little is however known about the properties of T lymphocytes in chronic pancreatitis.
Jon A. Tsai, MD, PhD Karolinska University Hospital Huddinge Department of Surgery K53 SE–141 86 Stockholm (Sweden) Tel. +46 8 58580000, Fax +46 8 58582340, E-Mail [email protected]
Table 1. Clinical characterization of nonresected and resected patients with chronic pancreatitis
Patient
Age
Gender
Etiology
Extent of Ongoing disease alcohol abuse
Weight loss
Diabetes
Nonresected A 47 B 48 C 52 D 58 E 62 F 64 G 66 H 66 I 75
M M F F F F M M M
Alcohol Alcohol Idiopathic Alcohol Alcohol Idiopathic Idiopathic Alcohol Idiopathic
Focal Diffuse Diffuse Diffuse Diffuse Diffuse Focal Focal Diffuse
No No No Yes Yes No No No Yes
No No Yes No Yes Yes No No Yes
Patient
Gender
Etiology
Extent of Indication PostSymptom disease for operative relief after surgery complications surgery
Time between Ongoing surgery and alcohol study, years abuse
Weight loss
Diabetes
M F M M M F M M F
Idiopathic Alcohol Idiopathic Alcohol Alcohol Idiopathic Alcohol Alcohol Idiopathic
Diffuse Focal Focal Diffuse Diffuse Diffuse Diffuse Focal Focal
2.5 3 2.5 2 0.5 2.5 2 2.5 1
No Yes No No Yes No No No No
Yes Yes Yes No Yes No No No No
Age
Resected K 26 L 46 M 48 N 54 O 57 P 58 Q 62 R 67 S 68
No Yes NA No No NA NA No NA
Pain Tumor Pain Pain Pain Pain Pain Tumor Pain
None None None None None None None None Sepsis
Good Moderate Moderate Good Good Good Good Good Poor
NA Yes NA No No NA No Yes NA
NA = Not applicable.
In recent years, our knowledge about different subtypes of T lymphocytes has been substantially improved [12–18]. T lymphocytes are heterogeneous and comprise distinct populations that can be distinguished based on surface markers and effector functions such as cytokine secretion and cytotoxicity. In short, naïve T lymphocytes, which travel to secondary lymphoid organs in search of antigens, express the chemokine receptor CCR7 and the membrane-bound signalling protein CD45RA. After exposure to antigens and cytokines such as IL-2, IL-7 and IL-15 the naïve T lymphocytes differentiate into memory T lymphocytes. These can be divided into CCR7+/ CD45RA– central memory T lymphocytes, CCR7–/ CD45RA– effector memory T lymphocytes and for the CD8+ population, CCR7–/CD45RA+ terminal effector memory T lymphocytes [19, 20]. In rheumatoid arthritis, one study has shown a decrease of CD45RA+/CD62L–/CD8+ effector memory T lymphocytes and an increase of CD45RA–/CD62L+/ CD8+ central memory T lymphocytes in peripheral blood
178
Pancreatology 2005;5:177–182
of patients compared to healthy controls [21]. The synovial tissue of rheumatoid arthritis patients has an increase in naïve cells expressing CD45RA [22]. In contrast to these findings, another study of patients with autoimmune thyroid disease shows a decrease of CCR7+ naïve and central memory cells in peripheral blood [23]. In multiple sclerosis, an expansion of both CD4+ and CD8+ subsets with a memory phenotype was seen in the cerebrospinal fluid as compared to peripheral blood of the patients [24], and in peripheral blood a higher frequency of effector memory CD4+ T lymphocytes has been reported [25]. There is still a great need for further studies on the distribution of T lymphocyte subtypes in inflammatory diseases and the possible role of skewed T lymphocyte homeostasis in the inflammatory pathogenesis. Given the elusive pathology of chronic pancreatitis and the importance of T lymphocytes for chronic inflammation, we have analyzed peripheral blood mononuclear cells for different T lymphocyte subsets in patients with this disease.
Grundsten/Liu/Permert/Hjelmstrom/Tsai
Materials and Methods Patient Characterization The patients in this study had been referred to our clinic due to symptomatic chronic pancreatitis and were selected consecutively. In all cases, the diagnosis was confirmed by pancreatography that demonstrated dilatation of the pancreatic duct and computer tomography, which showed an inflammatory mass and/or calcifications in the pancreatic parenchyma. In the patients who were resected, the diagnosis was also confirmed by histopathological analysis of the specimen. Nine patients with chronic pancreatitis who had not been resected (age 59.8 8 3.1 years, 2.2 8 0.5 years time since onset of symptoms) (table 1), 9 patients who had undergone pancreatic resection due to chronic pancreatitis (age 53.4 8 4.1, 4.8 8 1.4 years of symptoms before surgery) ( table 1) and 9 healthy controls (mean age 50.3 8 4.7 years, 4 males/5 females) were included in the study. There was no significant age difference between the three groups (p = 0.26). The blood samples were taken when the patients were at the ambulatory ward, except in one case (patient B, who was hospitalized due to abdominal pain, without any signs of acute pancreatitis). A conservative approach was chosen for the patients in the nonresected group due to less severe symptoms and no suspicion of pancreatic cancer. At the time of collecting blood samples, the nonresected and the resected patients were fairly comparable in terms of gender, etiology (alcohol or idiopathic), extent of disease (focal or diffuse), ongoing alcohol abuse, weight loss and diabetes (table 1). All diabetic patients were on insulin treatment. The outcome of surgery in the resected group (good, fair, poor) is also given in table 1. One of the resected cases (patient S) had a postoperative complication (sepsis), this patient also used aspirin occasionally. The other patients in this study were not using corticosteroids or nonsteroidal anti-inflammatory drugs. The ethical committee at Karolinska Institutet approved this study. Flow Cytometry Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with Lymphoprep (Nycomed, Oslo, Norway) from heparinized blood specimens using standard protocol. The PBMCs of the patients and control subjects were characterized for the expression of chemokine receptors by three-color direct immunofluorescence and flow cytometry using a FACscan (Becton Dickinson). Cells were washed with PBS (0.5% BSA, pH 7.2) and resuspended in PBS at a final concentration of 4 ! 106 cell/ml. Cells used for staining with antibody were first Fc-blocked by treatment with 1 g of normal mouse IgG (Caltag Laboratories, Calif., USA) per 105 cells for 15 min at room temperature. The following antibodies were added with the Fc-blocked 1 ! 105 cells in 25 l cell suspension according to the direction of the manufacturer: (1) IgG1 FITC (B.D., Calif., USA), IgG2a PE (R&D Systems, UK), CD4 PerCP (B.D.); (2) CD45RA FITC (B.D.), CCR7 FITC (R&D Systems), CD4 PerCP; (3) IgG1 FITC, IgG2a PE, CD8 PerCP; (4) CD45RA FITC, CCR7 PE, CD8 PerCP (B.D.). After incubation for 30 min at 4 ° C, the cells were washed twice with PBS, fixed with 1% paraformaldehyde in PBS and analyzed on the FACscan using CellQuest software (Becton Dickinson).
Table 2. Subsets of CD4+ and CD8+ T lymphocytes in healthy
controls, nonresected and resected patients with chronic pancreatitis Controls (n = 9), %
CD 4+ Naïve Central memory Effector memory CD 8+ Naïve Central memory Effector memory Terminal effector
Nonresected Resected (n = 9), % (n = 9), %
p value (ANOVA)
5485.6 3485.1* 3486.1* 0.04 3285.7 5084.4* 4884.4* 0.03 8.081.5 13.782.9 10.981.8 0.18 3886.1 2484.4 2685.1 6.582.3 14.982.8* 16.782.0* 11.583.0 16.182.4 14.582.8 4486.5 4585.0 4384.2
0.16 0.01 0.49 0.95
* p < 0.05 for post-hoc comparison with healthy controls.
tine methods. The normal ranges of the different parameters are given in table 2. Interleukin-15 Plasma-EDTA was collected at the same time as samples for flow cytometry from the patients with chronic pancreatitis, except in one of the resected patients. The samples were centrifuged at 2,500 rpm for 10 min and the supernatants were stored at –70 ° C for later analysis with a commercial ELISA (detection limit 2 pg/ml) according to the instructions from the manufacturer (R&D Systems Inc., Minn., USA). Plasma samples from the controls used in the flow cytometry studies were not available. Instead, we measured plasma from 9 additional healthy controls with a similar age (46.6 8 2.5 years). Statistics The data is presented as mean 8 standard error of the mean (SEM) or median with range (IL-15). Data with a normal distribution was analyzed with one-way analysis of variance (ANOVA) with Student-Newman-Keul’s post-hoc test or Student’s t test in cases with two groups. Data with a non-normal distribution was analyzed with Mann-Whitney Rank Sum test or ANOVA on ranks. p values less than 0.05 were considered to be statistically significant. All calculations were performed with Sigma Stat software (Jandel Corp., Sausalito, Calif., USA).
Results
C-Reactive Protein and Leukocytes C-reactive protein (CRP) and leukocyte counts, including differential analysis, were determined in the Department of Clinical Chemistry at Karolinska University Hospital Huddinge, using rou-
T Lymphocyte Subsets Both nonresected and resected patients with chronic pancreatitis had significantly higher levels of central memory T lymphocytes (CCR7+/CD45RA–) compared to healthy controls (table 2). CD4+ naïve T lymphocytes (CCR7+/CD45RA+) were significantly lower in both groups of chronic pancreatitis patients and there was also
Memory T Cells in Chronic Pancreatitis
Pancreatology 2005;5:177–182
179
Table 3. Inflammatory parameters and distribution of CD4+ and CD8+ cells in nonresected and resected patients with chronic pancreatitis
Leukocytes, 109/l Neutrophils, 109/l Lymphocytes, 109/l Monocytes, 109/l Eosinophils, 109/l Basophils, 109/l CD4+, % CD8+, % 1
IL-15 (log pg/ml)
Nonresected (n = 9)
Resected (n = 9)
p value
4.0410.0 1.647.5 1.044.0 0.140.9 0.040.5 0.040.1 4683.21 2682.91
8.181.2 4.780.94 2.480.21 0.5580.14 0.4180.08 0.0580.009 4983.1 2983.6
8.480.76 4.880.67 2.880.37 0.5080.048 0.2080.028 0.0580.006 4782.5 2981.9
0.85 (t test) 0.91 (t test) 0.42 (t test) 0.76 (t test) 0.02 (t test) 0.85 (t test) 0.65 (ANOVA) 0.61 (ANOVA)
Mean 8 SEM in healthy controls (n = 9).
cantly higher in the nonresected patients compared to the resected patients (table 3). There was no difference in the levels of CD4+ cells and CD8+ cells between healthy controls and nonresected or resected patients (table 3).
1,000
100
10 Detection limit 1
0.1
Normal range
Controls Nonresected Resected
Plasma IL-15 There were no significant differences in plasma IL15 levels between the three groups (ANOVA on ranks): 19.2 pg/ml (median, range !2 to 287, n = 9) in healthy controls, 16.8 pg/ml (median, range !2 to 69.7 pg/ml, n = 9) in nonresected patients and 18.6 pg/ml (median, range !7.12 to 126, n = 8) in resected patients (fig. 1).
Fig. 1. Plasma IL-15 in healthy controls (n = 9), nonresected (n =
9) and resected patients (n = 8) with chronic pancreatitis. Horizontal bars indicate medians. There was no difference between the three groups (ANOVA on ranks).
a trend to lower CD8+ naïve cells compared to controls (table 2). There was no difference in effector memory or terminal effector T cells between the three groups. Leukocytes, CRP and Total CD4+ and CD8+ Cells Total leukocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils in both nonresected and resected patients with chronic pancreatitis were well within the normal ranges of the Hospital Laboratory (table 3). Two of the nonresected patients with chronic pancreatitis had a minor increase of CRP (13 and 46 ng/ml, respectively), while the remaining subjects in both patient groups had normal CRP levels (!10 ng/ml). The CRP levels in the two groups were not significantly different (p = 0.44, Mann-Whitney test). Eosinophils were signifi-
180
Pancreatology 2005;5:177–182
Discussion
This study shows that patients with chronic pancreatitis seem to have a skewed distribution of T lymphocyte subsets, with higher levels of central memory T lymphocytes and lower levels of naïve cells (only significant for CD4+ cells) compared to healthy controls. Lymphocyte counts were within the normal range and the distribution of total CD4+ and CD8+ cells in chronic pancreatitis patients was similar compared to healthy controls. This indicates that the increase of central memory T lymphocytes occurs due to an increased differentiation of naïve cells, rather than due to an isolated expansion of the central memory cell pool. The distribution of T lymphocytes in patients who had undergone pancreatic resection and patients who had not been resected was similar. The T lymphocyte subsets were not studied prospectively in the same patients, but the nonresected and resected groups were fairly similar in terms of disease characteristics, besides that the resected patients had a longer disease duration, more pain or a suspected pancreatic cancer preop-
Grundsten/Liu/Permert/Hjelmstrom/Tsai
eratively. Even after resection of the most pronounced area of inflammation in chronic pancreatitis that causes the greater part of the symptoms, usually the pancreatic head, there is certainly at least some degree of inflammation in the tissue that is not removed. Since the altered distribution of memory T cells was similar in nonresected and resected patients, it is possible that the remaining inflammatory tissue is sufficient to drive this immunologic dysregulation. Another possibility is that this systemic immunologic condition can persist regardless of the inflammatory status in the pancreas. In addition to the chronic pancreatitis, other factors that may have influenced the immunologic status of the patients have to be considered. Malnutrition, ongoing alcohol abuse and diabetes with insulin treatment, which all can have an influence on the immune system, were present in some of the patients. Alcohol abuse [26] and malnutrition [27] are associated with a downregulation of the immune system and an impaired defence against infections. Thus, it seems unlikely that these conditions should induce increased differentiation of memory T cells. In contrast, insulin treatment may have a stimulatory effect on lymphocytes [28], where an upregulation of insulin receptors occurs during activation of the cells [29]. The patients with chronic pancreatitis who were included in the present study had insulin treatment due to loss of pancreatic function and not because of insulin resistance, as in type II diabetes. Therefore, there should not have been any substantial hyperinsulinemia in these patients leading to an abnormal insulin-induced stimulation of lymphocytes. Still, we cannot exclude the possibility that alcohol abuse, malnutrition, insulin treatment or other unknown factors may have affected memory T cells, in addition to the chronic pancreatitis. The exact role of central memory T lymphocytes in human inflammatory diseases or in the development of inflammatory diseases in animal models is largely unknown. We can only speculate about what induces the increase in central memory T lymphocytes in chronic pancreatitis and what function these cells have in the development of the disease. An upregulation of specific cytokines, such as IL-15, during episodes of acute inflammation in the pancreas, which drive the differentiation of naïve cells to central memory [20] is a possible theory. There were no differences in IL-15 between controls, nonresected and resected patients, but these data do not rule out that IL-15 or other cytokines may be upregulated during early, asymptomatic stages of chronic pancreatitis. Genetic factors associated with chronic pancreatitis [30] or other factors yet to be characterized could also contrib-
ute to facilitate the differentiation of memory T cells. We also suggest that the skewed distribution of central memory T lymphocytes may be a mechanism that contributes to the maintenence of chronic pancreatitis, which persists also after the initial cause of the disease is eliminated, e.g. alcohol abuse. This immunologic derangement could be a general phenomenon in chronic inflammatory disorders, since upregulation of memory T cells has been described in other inflammatory conditions also [21, 24]. In contrast to previous studies, we found no differences in the distribution of total CD4+ and CD8+ cells between healthy controls, nonresected or resected chronic pancreatitis patients. Different patient characteristics, due to the heterogeneity of chronic pancreatitis, may have contributed to this discrepancy. In conclusion, this study shows that central memory T lymphocytes are upregulated in patients with chronic pancreatitis. This may reflect a persistent chronic inflammatory response that may have been induced during early stages of the disease. We speculate that this derangement may be important for maintaining the chronic inflammation. Further studies on the influence of different etiologies of chronic pancreatitis, other inflammatory diseases and subject age on the distribution of central memory cells are needed to gain more insights into the pathophysiological significance of this T lymphocyte subset.
Memory T Cells in Chronic Pancreatitis
Pancreatology 2005;5:177–182
Acknowledgments This study was supported by grants from the Swedish Society of Medicine and the Foundation of Magnus Bergwall.
181
References 1 Büchler MW, Friess H, Uhl W, Malfertheiner P: Chronic Pancreatitis – Novel Concepts in Biology and Therapy. Oxford, Blackwell Publishing, 2002. 2 Friess H, Büchler MW, Mueller C, Malfertheiner P: Immunopathogenesis of chronic pancreatitis. Gastroenterology 1998; 115: 1018– 1022. 3 Bedossa P, Bacci J, Lemaigre G, Martin E: Lymphocyte subsets and HLA-DR expression in normal pancreas and chronic pancreatitis. Pancreas 1990;5:415–420. 4 Ebert MP, Ademmer K, Muller-Ostermeyer F, Friess H, Büchler MW, Schubert W, Malfertheiner P: CD8+CD103+ T cells analogous to intestinal intraepithelial lymphocytes infiltrate the pancreas in chronic pancreatitis. Am J Gastroenterol 1998;93:2141–2147. 5 Emmrich J, Weber I, Nausch M, Sparmann G, Koch K, Seyfarth M, Lohr M, Liebe S: Immunohistochemical characterization of the pancreatic cellular infiltrate in normal pancreas, chronic pancreatitis and pancreatic carcinoma. Digestion 1998;59:192–198. 6 Hunger RE, Mueller C, Z’graggen K, Friess H, Büchler MW: Cytotoxic cells are activated in cellular infiltrates of alcoholic chronic pancreatitis. Gastroenterology 1997; 112: 1656– 1663. 7 Saurer L, Reber P, Schaffner T, Büchler MW, Buri C, Kappeler A, Walz A, Friess H, Mueller C: Differential expression of chemokines in normal pancreas and in chronic pancreatitis. Gastroenterology 2000;118:356–367. 8 Antal L, Kavai M, Szabo G, Sonkoly I, Paloczi K, Szegedi G, Sapy P, Varhelyi I: Immunological investigations in acute and chronic human pancreatitis. Digestion 1980;20:100–105. 9 Skuia NA, Veksler K, Berzinia SE: T-lymphocyte subpopulations in chronic pancreatitis. Ter Arkh 1987;59:84–87. 10 Ockenga J, Jacobs R, Kemper A, Benschop RJ, Schmidt RE, Manns MP: Lymphocyte subsets and cellular immunity in patients with chronic pancreatitis. Digestion 2000;62:14–21.
182
11 Gansauge F, Gansauge S, Eh M, Schlosser W, Ramadani M, Kern P, Beger HG: Distributional and functional alterations of immunocompetent peripheral blood lymphocytes in patients with chronic pancreatitis. Ann Surg 2001;233:365–370. 12 Baron V, Bouneaud C, Cumano A, Lim A, Arstila TP, Kourilsky P, Ferradini L, Pannetier C: The repertoires of circulating human CD8(+) central and effector memory T cell subsets are largely distinct. Immunity 2003;18: 193–204. 13 Geginat J, Sallusto F, Lanzavecchia A: Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells. J Exp Med 2001;194: 1711–1719. 14 Geginat J, Lanzavecchia A, Sallusto F: Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines. Blood 2003; 101:4260–4266. 15 Hamann D, Baars PA, Rep MH, Hooibrink KB, Kerkhof-Garde SR, Klein MR, van Lier RA: Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med 1997;186:1407–1418. 16 Masopust D, Vezys V, Marzo AL, Lefrancois L: Preferential localization of effector memory cells in nonlymphoid tissue. Science 2001;291: 2413–2417. 17 Reinhardt RL, Khoruts A, Merica R, Zell T, Jenkins MK: Visualizing the generation of memory CD4 T cells in the whole body. Nature 2001;410:101–105. 18 Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A: Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999;401:708–712. 19 Farber DL: Remembrance of antigens past: New insights into memory T cells. Scand J Immunol 2003;58:145–154. 20 Schluns KS, Lefrancois L: Cytokine control of memory T-cell development and survival. Nat Rev Immunol 2003;3:269–279. 21 Maldonado A, Mueller YM, Thomas P, Bojczuk P, O’Connors C, Katsikis PD: Decreased effector memory CD45RA+ CD62L– CD8+ T cells and increased central memory CD45RA– CD62L+ CD8+ T cells in peripheral blood of rheumatoid arthritis patients. Arthritis Res Ther 2003;5:R91–R96.
Pancreatology 2005;5:177–182
22 Weninger W, Carlsen HS, Goodarzi M, Moazed F, Crowley MA, Baekkevold ES, Cavanagh LL, von Andrian UH: Naive T cell recruitment to nonlymphoid tissues: A role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis. J Immunol 2003;170:4638–4648. 23 Armengol MP, Cardoso-Schmidt CB, Fernandez M, Ferrer X, Pujol-Borrell R, Juan M: Chemokines determine local lymphoneogenesis and a reduction of circulating CXCR4(+) T and CCR7 B and T lymphocytes in thyroid autoimmune diseases. J Immunol 2003; 170: 6320–6328. 24 Giunti D, Borsellino G, Benelli R, Marchese M, Capello E, Valle MT, Pedemonte E, Noonan D, Albini A, Bernardi G, Mancardi GL, Battistini L, Uccelli A: Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS. J Leukoc Biol 2003;73:584–590. 25 Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, Chandy KD: The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. J Clin Invest 2003;111:1703–1713. 26 Szabo G: Consequences of alcohol consumption on host defence. Alcohol Alcohol 1999;34: 830–841. 27 Savino W: The thymus gland is a target in malnutrition. Eur J Clin Nutr 2002; 56(suppl 3): S46–S49. 28 Schloot NC, Roep BO, Wegmann D, Yu L, Chase HP, Wang T, Eisenbarth GS: Altered immune response to insulin in newly diagnosed compared to insulin-treated diabetic patients and healthy control subjects. Diabetologia 1997;40:564–572. 29 Stentz FB, Kitabchi AE: De novo emergence of growth factor receptors in activated human CD4+ and CD8+ T lymphocytes. Metabolism 2004;53:117–122. 30 Grendell JH: Genetic factors in pancreatitis. Curr Gastroenterol Rep 2003;5:105–109.
Grundsten/Liu/Permert/Hjelmstrom/Tsai
Pancreatology 2005;5:177–182 DOI: 10.1159/000085269
Increased Central Memory T Cells in Patients with Chronic Pancreatitis Måns Grundstena Guang-Zhi Liub Johan Permerta Peter Hjelmstromb Jon A. Tsaia a Center for Surgical Sciences, Department of Surgery, and b Department of Neurotec, Division of Neurology, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden
Key Words Chronic pancreatitis Central memory T cells CCR7 CD45RA Pancreatic resection
flammatory site. We suggest that the increase of central memory T lymphocytes may be important for maintaining the inflammatory process in chronic pancreatitis. Copyright © 2005 S. Karger AG, Basel and IAP
Abstract Background/Aims: A dysregulated immune response has been suggested to be important for the pathogenesis of chronic pancreatitis (CP). Formation of immunological memory is based on the differentiation of naïve T lymphocytes to memory T lymphocytes after exposure to antigens and specific cytokines. The aim of this study was to analyze peripheral blood mononuclear cells (PBMCs) in patients with CP for different T lymphocyte subsets including naïve and memory T cells. Methods: PBMCs from 9 patients who had undergone pancreatic resection due to CP, 9 CP patients who had not been resected and 9 healthy controls were analyzed by flow cytometry. Results: Patients with CP had a skewed distribution of T lymphocytes, with an increased level of CCR7+/CD45RA– central memory T lymphocytes compared to healthy controls. Nonresected CP patients and subjects who had undergone pancreatic resection due to CP had similar levels of central memory T lymphocytes. Conclusion: Our results indicate that the dysregulation of the immune system in chronic pancreatitis seems to persist even after removal of large parts of the local in-
© 2005 S. Karger AG, Basel and IAP 1424–3903/05/0053–0177$22.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
Introduction
Chronic pancreatitis is a disease charaterized by painful progressive destruction of the pancreatic gland with loss of its exocrine and endocrine functions [1]. The pathophysiological mechanisms are not fully understood, although components of the immune system have been implicated as key elements in the disease [2]. The pancreas is infiltrated by lymphocytes in chronic pancreatitis [3–6] and this appears to at least partially be mediated by upregulation of chemokines in the inflamed gland [7]. In the peripheral blood of chronic pancreatitis patients, several studies have shown a dysregulated distribution of lymphocytes that in some cases is reversed after pancreatic resection [8–11]. T lymphocytes appear to play a key role in the inflammatory process in chronic pancreatitis and constitute a large part of both the cell population infiltrating the pancreas and the dysregulated cell population in peripheral blood. Little is however known about the properties of T lymphocytes in chronic pancreatitis.
Jon A. Tsai, MD, PhD Karolinska University Hospital Huddinge Department of Surgery K53 SE–141 86 Stockholm (Sweden) Tel. +46 8 58580000, Fax +46 8 58582340, E-Mail [email protected]
Table 1. Clinical characterization of nonresected and resected patients with chronic pancreatitis
Patient
Age
Gender
Etiology
Extent of Ongoing disease alcohol abuse
Weight loss
Diabetes
Nonresected A 47 B 48 C 52 D 58 E 62 F 64 G 66 H 66 I 75
M M F F F F M M M
Alcohol Alcohol Idiopathic Alcohol Alcohol Idiopathic Idiopathic Alcohol Idiopathic
Focal Diffuse Diffuse Diffuse Diffuse Diffuse Focal Focal Diffuse
No No No Yes Yes No No No Yes
No No Yes No Yes Yes No No Yes
Patient
Gender
Etiology
Extent of Indication PostSymptom disease for operative relief after surgery complications surgery
Time between Ongoing surgery and alcohol study, years abuse
Weight loss
Diabetes
M F M M M F M M F
Idiopathic Alcohol Idiopathic Alcohol Alcohol Idiopathic Alcohol Alcohol Idiopathic
Diffuse Focal Focal Diffuse Diffuse Diffuse Diffuse Focal Focal
2.5 3 2.5 2 0.5 2.5 2 2.5 1
No Yes No No Yes No No No No
Yes Yes Yes No Yes No No No No
Age
Resected K 26 L 46 M 48 N 54 O 57 P 58 Q 62 R 67 S 68
No Yes NA No No NA NA No NA
Pain Tumor Pain Pain Pain Pain Pain Tumor Pain
None None None None None None None None Sepsis
Good Moderate Moderate Good Good Good Good Good Poor
NA Yes NA No No NA No Yes NA
NA = Not applicable.
In recent years, our knowledge about different subtypes of T lymphocytes has been substantially improved [12–18]. T lymphocytes are heterogeneous and comprise distinct populations that can be distinguished based on surface markers and effector functions such as cytokine secretion and cytotoxicity. In short, naïve T lymphocytes, which travel to secondary lymphoid organs in search of antigens, express the chemokine receptor CCR7 and the membrane-bound signalling protein CD45RA. After exposure to antigens and cytokines such as IL-2, IL-7 and IL-15 the naïve T lymphocytes differentiate into memory T lymphocytes. These can be divided into CCR7+/ CD45RA– central memory T lymphocytes, CCR7–/ CD45RA– effector memory T lymphocytes and for the CD8+ population, CCR7–/CD45RA+ terminal effector memory T lymphocytes [19, 20]. In rheumatoid arthritis, one study has shown a decrease of CD45RA+/CD62L–/CD8+ effector memory T lymphocytes and an increase of CD45RA–/CD62L+/ CD8+ central memory T lymphocytes in peripheral blood
178
Pancreatology 2005;5:177–182
of patients compared to healthy controls [21]. The synovial tissue of rheumatoid arthritis patients has an increase in naïve cells expressing CD45RA [22]. In contrast to these findings, another study of patients with autoimmune thyroid disease shows a decrease of CCR7+ naïve and central memory cells in peripheral blood [23]. In multiple sclerosis, an expansion of both CD4+ and CD8+ subsets with a memory phenotype was seen in the cerebrospinal fluid as compared to peripheral blood of the patients [24], and in peripheral blood a higher frequency of effector memory CD4+ T lymphocytes has been reported [25]. There is still a great need for further studies on the distribution of T lymphocyte subtypes in inflammatory diseases and the possible role of skewed T lymphocyte homeostasis in the inflammatory pathogenesis. Given the elusive pathology of chronic pancreatitis and the importance of T lymphocytes for chronic inflammation, we have analyzed peripheral blood mononuclear cells for different T lymphocyte subsets in patients with this disease.
Grundsten/Liu/Permert/Hjelmstrom/Tsai
Materials and Methods Patient Characterization The patients in this study had been referred to our clinic due to symptomatic chronic pancreatitis and were selected consecutively. In all cases, the diagnosis was confirmed by pancreatography that demonstrated dilatation of the pancreatic duct and computer tomography, which showed an inflammatory mass and/or calcifications in the pancreatic parenchyma. In the patients who were resected, the diagnosis was also confirmed by histopathological analysis of the specimen. Nine patients with chronic pancreatitis who had not been resected (age 59.8 8 3.1 years, 2.2 8 0.5 years time since onset of symptoms) (table 1), 9 patients who had undergone pancreatic resection due to chronic pancreatitis (age 53.4 8 4.1, 4.8 8 1.4 years of symptoms before surgery) ( table 1) and 9 healthy controls (mean age 50.3 8 4.7 years, 4 males/5 females) were included in the study. There was no significant age difference between the three groups (p = 0.26). The blood samples were taken when the patients were at the ambulatory ward, except in one case (patient B, who was hospitalized due to abdominal pain, without any signs of acute pancreatitis). A conservative approach was chosen for the patients in the nonresected group due to less severe symptoms and no suspicion of pancreatic cancer. At the time of collecting blood samples, the nonresected and the resected patients were fairly comparable in terms of gender, etiology (alcohol or idiopathic), extent of disease (focal or diffuse), ongoing alcohol abuse, weight loss and diabetes (table 1). All diabetic patients were on insulin treatment. The outcome of surgery in the resected group (good, fair, poor) is also given in table 1. One of the resected cases (patient S) had a postoperative complication (sepsis), this patient also used aspirin occasionally. The other patients in this study were not using corticosteroids or nonsteroidal anti-inflammatory drugs. The ethical committee at Karolinska Institutet approved this study. Flow Cytometry Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with Lymphoprep (Nycomed, Oslo, Norway) from heparinized blood specimens using standard protocol. The PBMCs of the patients and control subjects were characterized for the expression of chemokine receptors by three-color direct immunofluorescence and flow cytometry using a FACscan (Becton Dickinson). Cells were washed with PBS (0.5% BSA, pH 7.2) and resuspended in PBS at a final concentration of 4 ! 106 cell/ml. Cells used for staining with antibody were first Fc-blocked by treatment with 1 g of normal mouse IgG (Caltag Laboratories, Calif., USA) per 105 cells for 15 min at room temperature. The following antibodies were added with the Fc-blocked 1 ! 105 cells in 25 l cell suspension according to the direction of the manufacturer: (1) IgG1 FITC (B.D., Calif., USA), IgG2a PE (R&D Systems, UK), CD4 PerCP (B.D.); (2) CD45RA FITC (B.D.), CCR7 FITC (R&D Systems), CD4 PerCP; (3) IgG1 FITC, IgG2a PE, CD8 PerCP; (4) CD45RA FITC, CCR7 PE, CD8 PerCP (B.D.). After incubation for 30 min at 4 ° C, the cells were washed twice with PBS, fixed with 1% paraformaldehyde in PBS and analyzed on the FACscan using CellQuest software (Becton Dickinson).
Table 2. Subsets of CD4+ and CD8+ T lymphocytes in healthy
controls, nonresected and resected patients with chronic pancreatitis Controls (n = 9), %
CD 4+ Naïve Central memory Effector memory CD 8+ Naïve Central memory Effector memory Terminal effector
Nonresected Resected (n = 9), % (n = 9), %
p value (ANOVA)
5485.6 3485.1* 3486.1* 0.04 3285.7 5084.4* 4884.4* 0.03 8.081.5 13.782.9 10.981.8 0.18 3886.1 2484.4 2685.1 6.582.3 14.982.8* 16.782.0* 11.583.0 16.182.4 14.582.8 4486.5 4585.0 4384.2
0.16 0.01 0.49 0.95
* p < 0.05 for post-hoc comparison with healthy controls.
tine methods. The normal ranges of the different parameters are given in table 2. Interleukin-15 Plasma-EDTA was collected at the same time as samples for flow cytometry from the patients with chronic pancreatitis, except in one of the resected patients. The samples were centrifuged at 2,500 rpm for 10 min and the supernatants were stored at –70 ° C for later analysis with a commercial ELISA (detection limit 2 pg/ml) according to the instructions from the manufacturer (R&D Systems Inc., Minn., USA). Plasma samples from the controls used in the flow cytometry studies were not available. Instead, we measured plasma from 9 additional healthy controls with a similar age (46.6 8 2.5 years). Statistics The data is presented as mean 8 standard error of the mean (SEM) or median with range (IL-15). Data with a normal distribution was analyzed with one-way analysis of variance (ANOVA) with Student-Newman-Keul’s post-hoc test or Student’s t test in cases with two groups. Data with a non-normal distribution was analyzed with Mann-Whitney Rank Sum test or ANOVA on ranks. p values less than 0.05 were considered to be statistically significant. All calculations were performed with Sigma Stat software (Jandel Corp., Sausalito, Calif., USA).
Results
C-Reactive Protein and Leukocytes C-reactive protein (CRP) and leukocyte counts, including differential analysis, were determined in the Department of Clinical Chemistry at Karolinska University Hospital Huddinge, using rou-
T Lymphocyte Subsets Both nonresected and resected patients with chronic pancreatitis had significantly higher levels of central memory T lymphocytes (CCR7+/CD45RA–) compared to healthy controls (table 2). CD4+ naïve T lymphocytes (CCR7+/CD45RA+) were significantly lower in both groups of chronic pancreatitis patients and there was also
Memory T Cells in Chronic Pancreatitis
Pancreatology 2005;5:177–182
179
Table 3. Inflammatory parameters and distribution of CD4+ and CD8+ cells in nonresected and resected patients with chronic pancreatitis
Leukocytes, 109/l Neutrophils, 109/l Lymphocytes, 109/l Monocytes, 109/l Eosinophils, 109/l Basophils, 109/l CD4+, % CD8+, % 1
IL-15 (log pg/ml)
Nonresected (n = 9)
Resected (n = 9)
p value
4.0410.0 1.647.5 1.044.0 0.140.9 0.040.5 0.040.1 4683.21 2682.91
8.181.2 4.780.94 2.480.21 0.5580.14 0.4180.08 0.0580.009 4983.1 2983.6
8.480.76 4.880.67 2.880.37 0.5080.048 0.2080.028 0.0580.006 4782.5 2981.9
0.85 (t test) 0.91 (t test) 0.42 (t test) 0.76 (t test) 0.02 (t test) 0.85 (t test) 0.65 (ANOVA) 0.61 (ANOVA)
Mean 8 SEM in healthy controls (n = 9).
cantly higher in the nonresected patients compared to the resected patients (table 3). There was no difference in the levels of CD4+ cells and CD8+ cells between healthy controls and nonresected or resected patients (table 3).
1,000
100
10 Detection limit 1
0.1
Normal range
Controls Nonresected Resected
Plasma IL-15 There were no significant differences in plasma IL15 levels between the three groups (ANOVA on ranks): 19.2 pg/ml (median, range !2 to 287, n = 9) in healthy controls, 16.8 pg/ml (median, range !2 to 69.7 pg/ml, n = 9) in nonresected patients and 18.6 pg/ml (median, range !7.12 to 126, n = 8) in resected patients (fig. 1).
Fig. 1. Plasma IL-15 in healthy controls (n = 9), nonresected (n =
9) and resected patients (n = 8) with chronic pancreatitis. Horizontal bars indicate medians. There was no difference between the three groups (ANOVA on ranks).
a trend to lower CD8+ naïve cells compared to controls (table 2). There was no difference in effector memory or terminal effector T cells between the three groups. Leukocytes, CRP and Total CD4+ and CD8+ Cells Total leukocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils in both nonresected and resected patients with chronic pancreatitis were well within the normal ranges of the Hospital Laboratory (table 3). Two of the nonresected patients with chronic pancreatitis had a minor increase of CRP (13 and 46 ng/ml, respectively), while the remaining subjects in both patient groups had normal CRP levels (!10 ng/ml). The CRP levels in the two groups were not significantly different (p = 0.44, Mann-Whitney test). Eosinophils were signifi-
180
Pancreatology 2005;5:177–182
Discussion
This study shows that patients with chronic pancreatitis seem to have a skewed distribution of T lymphocyte subsets, with higher levels of central memory T lymphocytes and lower levels of naïve cells (only significant for CD4+ cells) compared to healthy controls. Lymphocyte counts were within the normal range and the distribution of total CD4+ and CD8+ cells in chronic pancreatitis patients was similar compared to healthy controls. This indicates that the increase of central memory T lymphocytes occurs due to an increased differentiation of naïve cells, rather than due to an isolated expansion of the central memory cell pool. The distribution of T lymphocytes in patients who had undergone pancreatic resection and patients who had not been resected was similar. The T lymphocyte subsets were not studied prospectively in the same patients, but the nonresected and resected groups were fairly similar in terms of disease characteristics, besides that the resected patients had a longer disease duration, more pain or a suspected pancreatic cancer preop-
Grundsten/Liu/Permert/Hjelmstrom/Tsai
eratively. Even after resection of the most pronounced area of inflammation in chronic pancreatitis that causes the greater part of the symptoms, usually the pancreatic head, there is certainly at least some degree of inflammation in the tissue that is not removed. Since the altered distribution of memory T cells was similar in nonresected and resected patients, it is possible that the remaining inflammatory tissue is sufficient to drive this immunologic dysregulation. Another possibility is that this systemic immunologic condition can persist regardless of the inflammatory status in the pancreas. In addition to the chronic pancreatitis, other factors that may have influenced the immunologic status of the patients have to be considered. Malnutrition, ongoing alcohol abuse and diabetes with insulin treatment, which all can have an influence on the immune system, were present in some of the patients. Alcohol abuse [26] and malnutrition [27] are associated with a downregulation of the immune system and an impaired defence against infections. Thus, it seems unlikely that these conditions should induce increased differentiation of memory T cells. In contrast, insulin treatment may have a stimulatory effect on lymphocytes [28], where an upregulation of insulin receptors occurs during activation of the cells [29]. The patients with chronic pancreatitis who were included in the present study had insulin treatment due to loss of pancreatic function and not because of insulin resistance, as in type II diabetes. Therefore, there should not have been any substantial hyperinsulinemia in these patients leading to an abnormal insulin-induced stimulation of lymphocytes. Still, we cannot exclude the possibility that alcohol abuse, malnutrition, insulin treatment or other unknown factors may have affected memory T cells, in addition to the chronic pancreatitis. The exact role of central memory T lymphocytes in human inflammatory diseases or in the development of inflammatory diseases in animal models is largely unknown. We can only speculate about what induces the increase in central memory T lymphocytes in chronic pancreatitis and what function these cells have in the development of the disease. An upregulation of specific cytokines, such as IL-15, during episodes of acute inflammation in the pancreas, which drive the differentiation of naïve cells to central memory [20] is a possible theory. There were no differences in IL-15 between controls, nonresected and resected patients, but these data do not rule out that IL-15 or other cytokines may be upregulated during early, asymptomatic stages of chronic pancreatitis. Genetic factors associated with chronic pancreatitis [30] or other factors yet to be characterized could also contrib-
ute to facilitate the differentiation of memory T cells. We also suggest that the skewed distribution of central memory T lymphocytes may be a mechanism that contributes to the maintenence of chronic pancreatitis, which persists also after the initial cause of the disease is eliminated, e.g. alcohol abuse. This immunologic derangement could be a general phenomenon in chronic inflammatory disorders, since upregulation of memory T cells has been described in other inflammatory conditions also [21, 24]. In contrast to previous studies, we found no differences in the distribution of total CD4+ and CD8+ cells between healthy controls, nonresected or resected chronic pancreatitis patients. Different patient characteristics, due to the heterogeneity of chronic pancreatitis, may have contributed to this discrepancy. In conclusion, this study shows that central memory T lymphocytes are upregulated in patients with chronic pancreatitis. This may reflect a persistent chronic inflammatory response that may have been induced during early stages of the disease. We speculate that this derangement may be important for maintaining the chronic inflammation. Further studies on the influence of different etiologies of chronic pancreatitis, other inflammatory diseases and subject age on the distribution of central memory cells are needed to gain more insights into the pathophysiological significance of this T lymphocyte subset.
Memory T Cells in Chronic Pancreatitis
Pancreatology 2005;5:177–182
Acknowledgments This study was supported by grants from the Swedish Society of Medicine and the Foundation of Magnus Bergwall.
181
References 1 Büchler MW, Friess H, Uhl W, Malfertheiner P: Chronic Pancreatitis – Novel Concepts in Biology and Therapy. Oxford, Blackwell Publishing, 2002. 2 Friess H, Büchler MW, Mueller C, Malfertheiner P: Immunopathogenesis of chronic pancreatitis. Gastroenterology 1998; 115: 1018– 1022. 3 Bedossa P, Bacci J, Lemaigre G, Martin E: Lymphocyte subsets and HLA-DR expression in normal pancreas and chronic pancreatitis. Pancreas 1990;5:415–420. 4 Ebert MP, Ademmer K, Muller-Ostermeyer F, Friess H, Büchler MW, Schubert W, Malfertheiner P: CD8+CD103+ T cells analogous to intestinal intraepithelial lymphocytes infiltrate the pancreas in chronic pancreatitis. Am J Gastroenterol 1998;93:2141–2147. 5 Emmrich J, Weber I, Nausch M, Sparmann G, Koch K, Seyfarth M, Lohr M, Liebe S: Immunohistochemical characterization of the pancreatic cellular infiltrate in normal pancreas, chronic pancreatitis and pancreatic carcinoma. Digestion 1998;59:192–198. 6 Hunger RE, Mueller C, Z’graggen K, Friess H, Büchler MW: Cytotoxic cells are activated in cellular infiltrates of alcoholic chronic pancreatitis. Gastroenterology 1997; 112: 1656– 1663. 7 Saurer L, Reber P, Schaffner T, Büchler MW, Buri C, Kappeler A, Walz A, Friess H, Mueller C: Differential expression of chemokines in normal pancreas and in chronic pancreatitis. Gastroenterology 2000;118:356–367. 8 Antal L, Kavai M, Szabo G, Sonkoly I, Paloczi K, Szegedi G, Sapy P, Varhelyi I: Immunological investigations in acute and chronic human pancreatitis. Digestion 1980;20:100–105. 9 Skuia NA, Veksler K, Berzinia SE: T-lymphocyte subpopulations in chronic pancreatitis. Ter Arkh 1987;59:84–87. 10 Ockenga J, Jacobs R, Kemper A, Benschop RJ, Schmidt RE, Manns MP: Lymphocyte subsets and cellular immunity in patients with chronic pancreatitis. Digestion 2000;62:14–21.
182
11 Gansauge F, Gansauge S, Eh M, Schlosser W, Ramadani M, Kern P, Beger HG: Distributional and functional alterations of immunocompetent peripheral blood lymphocytes in patients with chronic pancreatitis. Ann Surg 2001;233:365–370. 12 Baron V, Bouneaud C, Cumano A, Lim A, Arstila TP, Kourilsky P, Ferradini L, Pannetier C: The repertoires of circulating human CD8(+) central and effector memory T cell subsets are largely distinct. Immunity 2003;18: 193–204. 13 Geginat J, Sallusto F, Lanzavecchia A: Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells. J Exp Med 2001;194: 1711–1719. 14 Geginat J, Lanzavecchia A, Sallusto F: Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines. Blood 2003; 101:4260–4266. 15 Hamann D, Baars PA, Rep MH, Hooibrink KB, Kerkhof-Garde SR, Klein MR, van Lier RA: Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med 1997;186:1407–1418. 16 Masopust D, Vezys V, Marzo AL, Lefrancois L: Preferential localization of effector memory cells in nonlymphoid tissue. Science 2001;291: 2413–2417. 17 Reinhardt RL, Khoruts A, Merica R, Zell T, Jenkins MK: Visualizing the generation of memory CD4 T cells in the whole body. Nature 2001;410:101–105. 18 Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A: Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999;401:708–712. 19 Farber DL: Remembrance of antigens past: New insights into memory T cells. Scand J Immunol 2003;58:145–154. 20 Schluns KS, Lefrancois L: Cytokine control of memory T-cell development and survival. Nat Rev Immunol 2003;3:269–279. 21 Maldonado A, Mueller YM, Thomas P, Bojczuk P, O’Connors C, Katsikis PD: Decreased effector memory CD45RA+ CD62L– CD8+ T cells and increased central memory CD45RA– CD62L+ CD8+ T cells in peripheral blood of rheumatoid arthritis patients. Arthritis Res Ther 2003;5:R91–R96.
Pancreatology 2005;5:177–182
22 Weninger W, Carlsen HS, Goodarzi M, Moazed F, Crowley MA, Baekkevold ES, Cavanagh LL, von Andrian UH: Naive T cell recruitment to nonlymphoid tissues: A role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis. J Immunol 2003;170:4638–4648. 23 Armengol MP, Cardoso-Schmidt CB, Fernandez M, Ferrer X, Pujol-Borrell R, Juan M: Chemokines determine local lymphoneogenesis and a reduction of circulating CXCR4(+) T and CCR7 B and T lymphocytes in thyroid autoimmune diseases. J Immunol 2003; 170: 6320–6328. 24 Giunti D, Borsellino G, Benelli R, Marchese M, Capello E, Valle MT, Pedemonte E, Noonan D, Albini A, Bernardi G, Mancardi GL, Battistini L, Uccelli A: Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS. J Leukoc Biol 2003;73:584–590. 25 Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, Chandy KD: The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. J Clin Invest 2003;111:1703–1713. 26 Szabo G: Consequences of alcohol consumption on host defence. Alcohol Alcohol 1999;34: 830–841. 27 Savino W: The thymus gland is a target in malnutrition. Eur J Clin Nutr 2002; 56(suppl 3): S46–S49. 28 Schloot NC, Roep BO, Wegmann D, Yu L, Chase HP, Wang T, Eisenbarth GS: Altered immune response to insulin in newly diagnosed compared to insulin-treated diabetic patients and healthy control subjects. Diabetologia 1997;40:564–572. 29 Stentz FB, Kitabchi AE: De novo emergence of growth factor receptors in activated human CD4+ and CD8+ T lymphocytes. Metabolism 2004;53:117–122. 30 Grendell JH: Genetic factors in pancreatitis. Curr Gastroenterol Rep 2003;5:105–109.
Grundsten/Liu/Permert/Hjelmstrom/Tsai