Comparison of Immunophenotypes in the Blood of Patients on Continuous

Advances in Peritoneal Dialysis, Vol. 18, 2002

Comparison of Immunophenotypes in the Blood of Patients on Continuous Ambulatory Peritoneal Dialysis, Asymptomatic and with Julio Kl nger,1,2 Jaime Enr quez,2,4 Jhon A. Arturo,1,2 Mario Delgado,3 Gloria Avila,1,2 Merrem Mosquera4 Peritonitis

Peritonitis is a complication of continuous ambulatory was below 1.2. Those patients also had more intense CD4 peritoneal dialysis (CAPD) that often causes fibrosis. lymphopenia, more CD8 cells (principally T-suppressor Understanding the role played by the immune system is cells), and more expansion of NK cells. required if we are to understand the mechanisms of In patients on CAPD, an important immune defense and tissue lesion triggered by germs. activation and rise in the percentage of B1 cells occurs We compared the characteristics of the blood that increases with peritonitis. Among the general immunophenotypes of patients on CAPD, with and clinical characteristics, albumin was the only one to without peritonitis. This descriptive, prospective study show a statistically significant difference between was carried out in the dialysis unit of San Jos University patients with and without peritonitis. An important CD4 Hospital, a tertiary care institution in Popay n, lymphopenia occurred in patients with a CD4:CD8 Colombia. ratio below 1.2. In blood samples from 46˚patients on CAPD (26˚with peritonitis and 20˚without peritonitis), we used Key words flow cytometry to measure cytokine production at the Immunophenotype, CAPD peritonitis, CD4:CD8 single-cell level. The diagnosis of peritonitis was made ratio, B1 cells by standard clinical and laboratory criteria. We noted general clinical characteristics of the patients; Introduction percentages of lymphocytes, monocytes, neutrophils, One solution for patients with kidney failure is and eosinophils; and cell counts of lymphocytes (CD4 continuous ambulatory peritoneal dialysis (CAPD), cells, CD8 cells) and their subsets [B1, B2, and natural whose most serious complications are peritonitis and killer (NK) cells]. We also determined the CD4:CD8 fibrosis, inducing failure of the dialysis technique ratio. (1,2). When germs enter the peritoneum, cells of the We found significant differences in the levels of serum immune system act in defense and trigger tissue injury albumin (p˚< 0.001), the percentages of lymphocytes (p˚< (3). The immune system is composed of two inter- 0.04) and neutrophils (p˚< 0.04), and the counts of B1 communicated cellular and molecular compartments and B2 cells, especially in patients whose CD4:CD8 ratio (3—5); those components are identified and isolated by flow cytometry, the staining of proteins specific for each cell group with fluorescent monoclonal From: 1Immunologic and Infectious Disease Investigations Laboratory, 2Nephrology Unit of the Department of Inter- antibodies called CD markers (6,7) nal Medicine, and 3Clinical Epidemiology Unit, Health Sciences Faculty, University of Cauca; and 4RTS Cauca— Immune system organization Nephrological, San Jos , Popay n, Colombia, South The immune system is composed of cells and America. molecules vigilantly defending and maintaining the 166 Kl nger et al homeostasis of the host. Functionally, the system has 35˚kDa from Glenodinium species (PerCP), human two branches: natural (innate, unspecific) immunity; immune-response D-related antigen (HLA-DR)]: anti- and specific (acquired) immunity. CD45 FITC/CD14 PE, CD4 FITC/CD3/PerCP/ Phagocytic cells manage innate immunity. Acquired HLA-DR PE, CD8 FITC/CD28 PE, CD5 FITC/CD19 immunity has two branches: humoral immunity PE, CD3 FITC/CD56 PE (Becton—Dickinson, San Jos , managed by B˚cells, which secrete antibodies or CA, U.S.A.). After incubation in the dark at 4°C for immunoglobulins; and cellular immunity managed by 30˚minutes, 2˚mL red-cell lysing solution (FACS: T˚cells, CD4+ and CD8+ (5). Becton—Dickinson) was added. After incubation at 37°C

Cells of the innate immune system (neutrophils, with 5% CO2 for 10˚minutes, the cells were centrifuged monocytes, eosinophils, and dendritic cells) start and at 1500˚rpm for 10˚minutes. After the supernatant was amplify the immune response by phagocytosis of germs removed, the cell pellet was washed twice in phosphate- and antigens, presenting them to T-helper (TH) CD4+ buffered saline (PBS). Finally, the pellet was diluted in cells from the specific immunity system. The T˚cells 0.5˚mL phosphate-buffered saline (PBS) and analyzed determine the kind of specific immunity that will fight in a FACSCalibur flow cytometer using the CellQuest the antigen: humoral or cellular (4,5). The B˚cells are software (Becton—Dickinson). CD19+, and two subsets exist: B1 cells [CD5+, fewer than 10% in peritoneum and 5% in blood; they produce Statistical analysis immunoglobulin˚M (IgM) and auto-antibodies], and B2 Analysis of the data was performed using STATA˚6.0 cells that produce the other classes of immunoglobulins. at the clinical epidemiology unit (INCLEN) of the The CD8 cells are divided into cytotoxics and Faculty of Health Sciences, University of Cauca. The suppressors, differentiated because the cytotoxics are data are provided as median and dispersion measures CD28+. Natural killer cells (NKs) are functionally from (range, standard deviation, and percentages). the innate system, but they are essential for inducing The data are kept in the clinical records and cellular immune responses (4—6). protected in fulfillment of the ethical principles of The objective of the present project was to observe research. The data generated from the project will be the presence and activation of the various populations useful for improving the health care of our patients of blood immune cells by flow cytometry in patients on CAPD. on CAPD with and without peritonitis. Results Patients and methods Table˚I shows the characteristics of both groups of Blood samples from 46˚patients on CAPD [26˚with patients. The mean ages and general profiles were peritonitis (group˚B) and 20˚without peritonitis similar. The groups contained more women than men. (group˚A)] were taken in the morning during the first The only parameter that was statistically significantly change of dialysis fluid. Patients were in the CAPD different was serum albumin. program of the San Jos Kidney Diseases Unit, CD4:CD8 ratio. The thinking behind that decision Department of Internal Medicine of the Health Sciences was that the CD4:CD8 ratio indicates either a decrease Faculty, University of Cauca, and the dialysis program of CD4+ cells or an expansion in CD8+ cells, and the of RTS Cauca in Popay n, Colombia. Dialysis observations of our lab associate a lower ratio with consisted of 4˚daily exchanges of Dianeal (Baxter the presence of chronic viruses such as those of the Healthcare Corporation, Deerfield, IL, U.S.A.). herpes group. The division was interesting, because The criteria for a diagnosis of peritonitis was the lower-ratio patients showed intense CD4 (A)˚cloudy dialysis fluid or abdominal pain or fever; lymphopenia, while having more cytotoxic cells (CD8 (B)˚>˚100˚leucocytes/mm3 in the fluid, with at least 50% and NK). neutrophils; and (C)˚detection of microbes by Gram High expression of HLA-DR, which was more stain or culture of the dialysis fluid. We exposed 50˚µL intense during peritonitis, was seen on T˚cells in CAPD blood to 20˚µL of each of the following fluorescent patients (Tables˚II and III; group˚A: 22.4%; group˚B: monoclonal antibodies [two or three antibodies per tube: 27.3%; healthy controls: >˚8%). fluorescein isothiocyanate conjugate (FITC), The percentage of CD8+CD28— cells (T-sup- phycoerythrin (PE), fluorescent chlorophilic protein of pressor cells) was high (<˚1.1) in all groups. Immunophenotypes in CAPD 167

TABLE I Characteristics of the studied population (n˚= 46)

Patients on continuous ambulatory peritoneal dialysis No peritonitis (n˚= 20) Peritonitis (n˚= 26) Parameter [n (%)] [n (%)] p Value

Mean age (years) 50.6 50.0 0.44 Sex Male 7 (35.0) 10 (38.5) Female 13 (65.0) 16 (61.5) 0.81 Provenance Urban 14 (70.0) 19 (73.1) 0.82 Rural 6 (30.0) 7 (26.9) Primary kidney disease 0.07 Diabetic nephropathy 5 (25.0) 13 (50.0) Chronic glomerulonephritis 5 (25.0) 9 (34.0) Others 10 (50.0) 4 (15.4) Previous peritonitis (mean episodes) 0.9 1.4 0.86 Mean serum albumin (g/dL) 3.6 3.0 0.001

TABLE II Characteristics of the immunophenotypes of patients on continuous ambulatory peritoneal dialysis (n˚= 46)

Patients on continuous ambulatory peritoneal dialysis Parameter No peritonitis (n˚= 20) Peritonitis (n˚= 26) p Value

Mean percentage of leucocytes Lymphocytes 17.7 13.5 0.04 Monocytes 6.7 6.1 0.51 Neutrophils 56.7 65.3 0.04 Eosinophils 5.4 5.7 0.90 Mean cell counts of CD4, CD8, B, and natural killer (NK) cells (/mm3) T CD4+ lymphocytes 528.8 475.6 0.61 T CD8+ lymphocytes 452.5 326.9 0.15 CD4:CD8 ratio 1.5 1.7 0.49 Percentages CD8+/CD28+ 34.2 42.8 0.06 CD8+/CD28— 65.8 57.2 0.32 HLA-DR on T˚cells 22.4 27.0 0.42 B1 CD5+/CD19+ cells 21.2 34.1 0.01 B2 CD5—/CD19— cells 78.8 65.9 0.01 NK cell expansion (>300˚cells/mm3) 0.20 Yes 11 (55%) 19 (73%) No 9 (45%) 7 (27%)

Expansion of NK cells was seen in 55% of Discussion patients of group˚A and in 73% of patients group˚B. The intense CD4 lymphopenia in patients with a That expansion was more intense in patients with lower CD4:CD8 ratio deserves further research to a low CD4:CD8 ratio and peritonitis (low-ratio look for its cause and clinical meaning. It could patients in group˚A: 40%; low-ratio patients in mean cellular immunodeficiency that could be group˚B: 78%). complicated with opportunistic infections and In both groups, total B˚cells were normal, but the tumors. percentage of B1 cells was high. The difference was Asymptomatic patients on CAPD showed more intense in the peritonitis group (Tables˚II important expression of HLA-DR on T˚lymphocytes, and˚III). which indicates immune activation. Those data accord 168 Kl nger et al

TABLE III Characteristics of the immunophenotypes of patients on continuous ambulatory peritoneal dialysis (n˚= 46) according to CD4:CD8 ratio

Patients on continuous ambulatory peritoneal dialysis CD4:CD8 ratio CD4:CD8 ratio Parameter <1.2 (n=10) ⊕1.2 (n=10) <1.2 (n=10) ⊕1.2 (n=17) p Value

Mean lymphocyte count (/mm3) T CD4+ lymphocytes 558 500 212.2 592 0.3 T CD8+ lymphocytes 676.8 228 397.6 295 0.28 Percentages CD8+/CD28+ lymphocytes 32.5 35.9 33 47.2 0.13 CD8+/CD28— lymphocytes 68.8 64 67 52 0.32 HLA-DR on T˚cells 29.6 15.2 26 23 0.26 B1 CD5+/CD19+ cells 22.8 20.1 34.7 33.9 0.01 B2 CD5—/CD19+ cells 77.2 79.9 65.2 66.1 0.01 with reports of immune activation in asymptomatic [macrophage colony-stimulating factor (M-CSF) and patients on CAPD, which is more intense during granulocyte colony-stimulating factor (G-CSF)]. peritonitis. Davies et al (8) saw increased chemi- Elevated levels of B1 cells in CAPD patients are luminescence on monocytes of asymptomatic patients enigmatic still, because of their greater rise during on CAPD, especially on peritoneal macrophages; high peritonitis. The obvious suggestion is that they expression of HLA-DR on T˚cells and monocytes; and produce IgM, the first immunoglobulin to peak after high expression of interleukin-2 receptor (IL-2R) and microbes gain entrance. immunoglobulin˚G (IgG) receptor. In another report, Lai et al (2) found a significant rise in neutrophil Conclusions numbers (400-fold) in dialysis fluid even 3˚weeks The present study, and the literature, found important after clinical resolution of peritonitis together with immune activation and increased secretion of increased numbers of dead mesothelial cells and high cytokines in asymptomatic CAPD patients. The production of pro-inflammatory cytokines and activation increases during peritonitis. Furthermore, transforming growth factor˚β (TGFβ). In parallel work we saw evidence of two different groups of patients in our laboratory, we found increased expression of according to the CD4:CD8 ratio. A lower ratio is CD69, HLA-DR, and pro-inflammatory cytokines in associated with more CD4 lymphopenia and more asymptomatic patients on CAPD, with an even greater activity of cytotoxic cells such as NK and rise during peritonitis. In sum, important elevation is CD8+CD28— cells. All of those findings suggest new seen in the numbers of immune activation markers in biologic and clinical research to explore the causes patients on CAPD, and the numbers increase even and influences of the immune system in the evolution more during peritonitis (9,10). and complications of patients on CAPD for example, It is important to recognize two groups of patients tissue degeneration (atherosclerosis, depression, and according to CD4:CD8 ratio, because the lower-ratio even aging, among others) (14,15). group is different. Patients in that group have more CD4 lymphopenia, especially during peritonitis; more References CD8 cells, especially the suppressor type; and more 1 Singh AK, Brenner BM. Dialysis in the treatment of NK cells. Those findings suggest a need for more renal failure. In: Braunwald E, Fauci AS, Kasper DL, research and care about the CD4:CD8 ratio in clinical et al, eds. Harrison s Principles of Internal Medicine. 15th ed. New York: McGraw—Hill; 2001: 1562—6. CAPD settings such as transplant (11). 2 Lai KN, Lai KB, Lam CW, Chan TM, Li FK, Leung The expansion of NK and CD8 cells accords with JC. Changes of cytokine profiles during peritonitis in reports from Saionji and Osaka (12) and Saionji et al patients on continuous ambulatory peritoneal dialy- (13), who found expansion of CD14+CD16+ cells in sis. Am J Kidney Dis 2000; 35:644—52. CAPD patients, along with high levels of factors that 3 Abbas AK, Lichtman AH, Pober JS. Propiedades stimulate monocyte and granulocyte production generales de las respuesta inmunitaria. In: Abbas AK, Immunophenotypes in CAPD 169

Lichtman AH, Pober JS, eds. Cellular and Molecular blood monocytes in patients with chronic renal Immunology. 3rd ed. Philadelphia: Saunders; 1997: failure undergoing dialysis: possible involvement of 1:3—15. macrophage colony-stimulating factor. Acta 4 Abbas AK, Lichtman AH, Pober JS. C lulas y tejidos Haematol 2001; 105:21—6. del sistema inmunitario. In: Abbas AK, Lichtman 13 Saionji K, Hamada T, Higurashi H, et al. Plasma AH, Pober JS, eds. Cellular and Molecular Immunol- macrophage colony-stimulating factor, granulocyte ogy. 3rd ed. Philadelphia: Saunders; 1997: 2:16—35. macrophage colony-stimulating factor and granulo- 5 Haynes BF, Fauci AS. Disorders of the immune cyte colony-stimulating factor levels in continuous system. In: Braunwald E, Fauci AS, Kasper DL, et al, ambulatory peritoneal dialysis patients. Rinsho Byori eds. Harrison s Principles of Internal Medicine. 15th 1997; 45:493—7. ed. New York: McGraw—Hill; 2001: 305:1805—30. 11 Marin GH, Menna ME, Saba S, et al. Flow cytomet- 6 Parks DR, Herzenberg LA. Flow cytometry and ric analysis of lymphocyte subsets as a predictive fluorescence-activated cell sorting. In: Paul WE, ed. factor for GVHD in allogeneic bone marrow trans- Fundamental immunology. New York: Raven Press; plantation. Transplant Proc 1999; 31:2973—5. 1989; 781—802. 14 Yudkin JS, Kumari M, Humphries SE, Mohame—Ali 7 Shapiro HM. Practical flow cytometry. 3rd ed. New V. Inflammation, obesity, stress and coronary heart York: Wiley—Liss; 1995: 1—517. disease: is interleukin-6 the link? Atherosclerosis 8 Davies SJ, Suassuna J, Ogg CS, Cameron JS. Activa- 2000; 148:209—14. tion of immunocompetent cells in the peritoneum of 15 Licinio J, Wong ML. The role of inflammatory me- patients treated with CAPD. Kidney Int 1988; 36: diators in the biology of major depression: central 661—8. nervous system cytokines modulate the biological 9 Pereira BJ. Cytokine production in patients on dialy- substrate of depressive symptoms, regulate stress- sis. Blood Purif 1995; 13:135—46. responsive systems, and contribute to neurotoxicity 10 Lu Y, Hylander B, Brauner A. Interleukin-10, inter- and neuroprotection. Mol Psychiatry 1999; feron gamma, interleukin-2, and soluble interleukin-2 4:317—27. receptor alpha detected during peritonitis in the dialysate and serum of patients on continuous ambulatory Corresponding author: peritoneal dialysis. Perit Dial Int 1996; 16:607—12. Julio Kl nger, MD, Calle˚17 Norte˚#14—16, Barrio 12 Saionji K, Ohsaka A. Expansion of CD4+CD16+ Machangara, Popay n—Cauca, Colombia, South America. Advances in Peritoneal Dialysis, Vol. 18, 2002

Cytokines and Peritonitis in Continuous Ambulatory Peritoneal Dialysis: Immunodeviation and Julio Kl nger,1 Jaime Enr quez,2,4 Jhon A. Arturo,1 Mario Delgado,3 Gloria Avila,1 Omaira Ceballos4 Immunodeficiency

Cytokines are soluble mediators of the immune system, TNFα, and IL-4, indicating in vivo immune activa- which regulate the immune response to antigenic tion similar to the group with peritonitis. In the group stimuli. In continuous ambulatory peritoneal dialysis without peritonitis, 95% of samples displayed (CAPD) patients with peritonitis, an inflammatory immunodeviation TH2. Just 5% of samples ap- process occurs, but the patterns of cytokine secretion proached TH0, producing IFN-γ. After mitogen acti- have not yet been clarified. We compared the vation, 45% of the samples stayed in TH2; 55% characteristics of the intracellular production of approached TH0. cytokines and looked for the immunophenotypes Patients with peritonitis produced high levels of T˚helper˚1 (TH1), T˚helper˚2 (TH2), and T˚helper˚0 IL-4 and little IFN-γ, which indicates immuno- (TH0) in CAPD patients with and without peritonitis. deviation TH2 in 75% of samples; 25% approached Our descriptive, prospective study was carried out in TH0. When cells were stimulated by ionomicin and the dialysis unit of the San Jos University Hospital, a phorbol 12-myristate 13-acetate (PMA), more IFN-γ tertiary health care center in Popay n, southwest appeared and high levels of IL-4 persisted in 75% of Colombia. the samples, which looked like intent to correct the We obtained 28 peripheral blood samples from TH2 immunodeviation toward TH0. CAPD patients (8˚with peritonitis and 20˚without Patients on CAPD with and without peritonitis peritonitis) and processed them by flow cytometry for showed immune activation per se and high production intracellular detection of cytokines. The peritonitis of pro-inflammatory cytokines accompanied by a diagnosis was made based using established clinical strong pattern of cytokine TH2 and a deficiency of and laboratory criteria. We measured the general IFN-γ production, suggesting heavy immunodeviation clinical characteristics and percentage of intracellular TH2 and immunodeficiency TH1 (owing to the deficit production of interleukin-1 (IL-1), interleukin-6 (IL-6), of IFN-γ). Finally, with in vitro immune stimulation, interleukin-12 (IL-12), tumor necrosis factor˚α the TH2 pattern tried to approach TH0. (TNFα), interleukin-4 (IL-4), and interferon-γ (INF-γ) in T˚lymphocytes. Key words Diabetic nephropathy and chronic glomerulo- Cytokines, TH1/TH2/TH0, immunoregulation, flow nephritis were the most frequent primary pathologies cytometry in both groups of patients. The patients on CAPD without peritonitis expressed high levels of CD69 and Introduction the pro-inflammatory cytokines IL-1, IL-6, IL-12, Continuous ambulatory peritoneal dialysis (CAPD) is a therapy that replaces the kidney function of patients with chronic renal insufficiency. The therapy From: 1Immunologic and Infectious Disease Investigations Laboratory, 2Nephrology Unit of the Department of Inter- is frequently complicated in some patients with nal Medicine, and 3Clinical Epidemiology Unit, Health repeated peritonitis, inducing peritoneal fibrosis and Sciences Faculty, University of Cauca; and 4RTS Cauca— irreversible loss of the dialysis capacity of peritoneum Nephrological, San Jos , Popay n, Colombia, South (1,2). Cytokines or interleukins (ILs) are proteins America. secreted during inflammation to modulate the immune Kl nger et al 171 response. In that way, cytokines influence healing and received 4˚daily exchanges of Dianeal (Baxter complications of infections (such as sepsis and tissue Healthcare Corporation, Deerfield, IL, U.S.A.). The fibrosis). Studying the behavior of cytokines is blood samples were taken during the years 2000˚— 2001. essential to understanding the mechanisms of defense All participants signed an informed consent to and complications in the war against germs (3—6). participate in the study. The data were collected using Cytokines are produced by many cell types. The an instrument that gathers sociodemographic and cytokines secreted by monocytes are called monokines, clinical information along with laboratory results. We and the ones secreted by lymphocytes are called decided to divide both groups of CAPD patients (with lymphokines. Monokines are pro-inflammatory and anti- and without peritonitis) into two subgroups, depending inflammatory (7,8), and the differences in the production on the value of the CD4:CD8 ratio (set point, 1.2), of cytokines by T-helper lymphocytes (TH CD4+) considering that low CD4:CD8 ratios are caused by a determine at least 3˚different subsets of T˚lymphocytes reduction of T CD4+ cells, and a predominance of T called TH1, TH2, and TH0 helper cells (9—11). CD8+ cells, as happens in infection by the human The TH1 lymphocytes are heavy producers of immunodeficiency virus (HIV) and as has been interferon-γ (IFN-γ) and IL-2. The TH2 subset observed in our laboratory in other chronic virus produces IL-4, IL-5, IL-6, IL-10, and IL-13. The TH0 infections such as cytomegalovirus, herpesvirus, and group secretes the entire range of TH1 and TH2 hepatitis B and C. cytokines. The TH lymphocyte subsets are important in the immunoregulation of the response to germs and Design antigens, and they influence the evolution of infectious Intracellular cytokines were detected by standard flow and immunologic diseases (10—13). The TH1 cells cytometry (13—16). induce cellular immunity, granuloma formation, and Cells were cultivated for 6˚hours with and without the delayed type of hypersensitivity. The TH2 subset mitogenic stimulation by phorbol 12-myristate favors production of the various immunoglobulin 13-acetate (PMA), ionomicin, and an inhibitor of the classes that shape or help humoral immunity. Golgi apparatus (Brefeldin˚A: Sigma, St.˚Louis, MO, An inflammatory process occurs in patients on U.S.A.; or GolgiStop: Becton—Dickinson, San Jos , CAPD with peritonitis, but the secretion patterns of CA, U.S.A.), which allows the intracellular cytokines has not yet been clarified (2,3). The objective accumulation of proteins. The proteins are then of the present study was to observe the intracellular detected by fluorescent monoclonal antibodies, production of the pro-inflammatory monokines IL-1, introduced after permeabilization of the cellular IL-6, IL-12, and tumor necrosis factor˚α (TNFα), and membrane with saponin (Cytofix/Cytoperm: Becton— the lymphokines IFN-γ and IL-4 in peripheral blood of Dickinson) (17). patients on CAPD with and without peritonitis. For intracellular staining, we used the following anti-cytokine antibodies conjugated with fluorescent Patients and methods substances [fluorescein isothiocyanate conjugate (FITC)/phycoerythrin (PE)/fluorescent chlorophilic Patients protein of 35˚kDa from Glenodinium species (PerCP)]: Peripheral blood samples were obtained from 20 anti-IL-1 FITC [mouse immunoglobulin˚G1 (IgG1), CAPD patients without peritonitis (group˚A) and 8 clone 364-3B3-14]˚/ IL-6 PE (rat IgG1, clone MQ2- CAPD patients who fulfilled at least two of the 1A5)˚/ CD3 PerCP, IL-12 FITC (mouse IgG1, clone following criteria for a diagnosis of peritonitis C11.5)˚/ TNFα PE (mouse IgG1, clone Mab11)˚/ CD3 (group˚B): (A)˚cloudy dialysate or abdominal pain or PerCP, and IL-4 FITC (rat IgG2b, clone BVD4- fever; (B)˚leukocyte count in peritoneal liquid greater 1D11)˚/ IFN-γ PE (rat IgG1, clone XMG1.2)˚/ CD3 than 100˚cells/mm3, with at least 50% neutrophils; PerCP (Pharmingen, San Diego, CA, U.S.A.). To (C)˚presence of micro-organisms by Gram stain or confirm cellular activation, we used anti-CD14 PE culture of dialysis liquid. (mouse IgG2b, clone MfP9), which identifies The patients came from the dialysis unit of San Jos monocytes by surface staining, and anti-CD69 (mouse University Hospital in Popay n, or the referral center IgG1, clone L78), an early marker of immunologic in the department of Cauca, southwest Colombia. All activation (Becton—Dickinson), in intracellular 172 Immunodeviation and Immunodeficiency in CAPD labeling. For identification of T-cell subsets and to Cellular permeabilization obtain the CD4:CD8 ratio, we used anti-CD8 FITC The cells were shaken in a vortex, and 250 µL (IgG1, clone SK1) and CD4 PE (mouse IgG1, clone permeabilization solution (Cytofix/Cytoperm: SK3). The antibodies for negative fluorescence control Becton—Dickinson), which contains saponin, was were R35-95 (rat IgG, 2APE) and MOPC-21 (mouse added to each tube. The tubes were incubated for IgG1) FITC (Pharmingen). 20˚minutes at 4°C in the dark. The cells were then washed twice, adding 1˚mL Perm/Wash (Becton— Procedure Dickinson) to each tube, and centrifuged at 500g for Culture and cellular activation were processed in two 5˚minutes. The supernatant was removed, leaving just parallel sets of tubes (with activation and without the pellet. activation). Whole blood (100˚µL) was dropped into the respective 12↔75˚mm polypropylene tubes, Intracellular staining together with 100˚µL RPMI-1640 complete cell The samples were diluted in 100˚µL Perm/Wash, and culture medium [containing 2˚mmol/L L-glutamine, 20˚xxL of each anti-cytokine antibody was add to the 250˚U/mL penicillin˚G, 250˚µg streptomycin (Gibco corresponding tubes, which were then incubated for BRL, Rockville, MD, U.S.A.), 50˚mmol/L of 30˚minutes at 4°C in the dark. The cells were then mercaptoethanol, and 10% bovine fetal serum]. Next, washed with 1˚mL PBS and centrifuged at 500g for 10˚µL Brefeldin˚A (stock solution, 1˚mg/mL) was 5˚minutes. After the supernatant was removed, the added to all tubes. To the activated tubes, we also cells were diluted with 500˚µL paraformaldehyde added 10˚µL ionomicin (stock solution, 1µg/mL) and (PFA, 1% solution) to fix the antibodies, the cells being 20˚µL phorbol 12-myristate 13-acetate (PMA: stock then ready for data acquisition in the flow cytometer. solution, 25˚ng/mL). The tubes were then incubated (The samples can be stored at 4°C in the dark for up ° for 6˚hours at 35 C in humidity and 5% CO2. to 24˚hours until data acquisition.) Data analysis was performed with the statistical Lysis of red cells package STATA˚6.0. The Student two-tailed t-test and After incubation, 2˚mL solution for lysing red blood the chi-square test were used to evaluate the differences cells (FACS: Becton—Dickinson) was added to all between the characteristics of the patients with and tubes. Incubation then continued for 10˚minutes at without peritonitis. A value of p˚< 0.05 was considered 37˚C. Afterward, the cells were centrifuged at statistically significant. 1500˚rpm for 10˚minutes, and the supernatant was removed. The cells were washed twice in 1˚mL phos- Results phate-buffered saline (PBS) and centrifuged for In the present study, 28˚patients on CAPD were in- 10˚minutes. cluded, as shown in Table˚I. The average of age of group˚A was 50.6˚years, and of group˚B, 45.5˚years. Blockade of Fc receptors In both groups, females predominated (65% and Soon after the second washing, the cell pellets were 75%). Diabetic nephropathy and chronic glomer- incubated with 50˚µL polyvalent human immuno- ulonephritis were the more frequent primary patholo- globulin˚G (Sandoglobulin: Novartis—Pharma AG, gies in both groups. Most of the patients came from Basle, Switzerland) for 20˚minutes in the dark at 4°C urban areas. No difference was seen in the number to block the Fc receptors and avoid unspecific bind- of previous episodes of peritonitis (p˚< 0.61). The ing of the antibodies that would later be used to dye average of the serum albumin levels was smaller in the cellular surface and the intracellular cytokines. group˚B (p˚< 0.001). Figure˚1 shows the percentage intracellular Surface staining production of cytokines in T˚lymphocytes (with no Antibodies (anti-CD3 PerCP, anti-CD4 PE, anti-CD4 PE, stimulation) in all of the CAPD patients. The group˚A anti-CD8 FITC, and anti-CD14: Becton—Dickinson) patients expressed high levels of the marker of early were added (20˚µL) to the corresponding tubes and activation (CD69) in monocytes and lymphocytes incubated for 30˚minutes at 4˚C in the dark. The cells 70% of cells (Data not shown). Group˚B patients were then washed once with PBS and centrifuged. showed similar results, suggesting immune activation Kl nger et al 173

TABLE I Characteristics of the continuous ambulatory peritoneal CAPD patients presented immunodeviation TH2; just dialysis patients with peritonitisa (n˚= 8) 5% approached TH0, producing IFN-γ. In group˚B patients, we observed high levels of Parameter N (%) production of pro-inflammatory cytokines and IL-4 Mean age (years) 45.5 (exceeding 89% of cells) and little IFN-γ (15.7% of Sex cells), indicating that 75% of those patients had Males 2 (25) immunodeviation TH2; 25% approached TH0. After Females 6 (75) stimulation in vitro (Figure˚2), cells from group˚A Provenance Urban 19 (73.1) showed persistent production of pro-inflammatory Rural 7 (26.9) cytokines and IL-4 at elevated levels, with a slight Renal disease increase in the production of IFN-γ in 55% of the Diabetic nephropathy 3 (37.5) patients (average: 36.4%). That finding indicates a Chronic glomerulonephritis 3 (37.5) tendency to correct the immunodeviation TH2 (45% Other glomerulopathies 2 (25.0) Previous mean episodes of peritonitis 0.62 of the patients) toward TH0 (55% of the patients). In Mean serum albumin (g/dL) 3.2 group˚B patients, the production of pro-inflammatory cytokines remained about the same, with the exception a The data for the 20 asymptomatic patients is similar (Not shown). of a slight reduction in TNFα. The IL-4 remained high, and better production of IFN-γ was observed, which in vivo or per se, that was increased with the mitogenic indicates a tendency to correct the immunodeviation stimuli, indicating success in the activation in vitro. TH2 toward TH0 in 75% of patients. In group˚A patients, we observed high levels of production of pro-inflammatory cytokines (IL-1, IL-6, Discussion IL-12, TNFα) that exceeded 80% of cells, with an The present study of CAPD patients with and without average of production of 92% in monocytes and lym- peritonitis made several important findings concerning phocytes (Table˚II). We also saw intense production the production of cytokines. Patients on CAPD without of IL-4 in T CD3+ lymphocytes (average: 92% of peritonitis presented intense immunologic activation cells); only 5% produced low levels of IFN-γ (aver- per se, expressing in vivo the early activation marker age: 4.8% of cells). Thus, 95% of the asymptomatic CD69 that has also been seen in other studies (18).

(A) (B) 100

90 100 80 90 70 IL-1 80 60 IL-6 70 IL-1 60 IL-6 50 IL-12 TNF-a 50 IL-12 40 IL-4 40 TNF-a 30 IFN-g 30 IL-4 IFN-g 20 20 10 10 0 0 Sin Con Sin Con Peritonitis Peritonitis Peritonitis Peritonitis

No Yes No Yes

FIGURE 1 (A)˚Intracellular cytokine production in the blood of 28 patients on continuous ambulatory peritoneal dialysis (8˚with peritonitis and 20 without peritonitis). No in vitro stimulation. (B)˚Intracellular cytokine production in the blood of the same 28˚patients, with in vitro stimulation. 174 Immunodeviation and Immunodeficiency in CAPD

TABLE II Comparison of the patterns T-helper˚1/T-helper˚2/T-helper˚0 (TH1/TH2/TH0) in patients on continuous ambulatory peritoneal dialysis with and without peritonitis.

Peritonitis No peritonitis CD4:CD8>1.2 CD4:CD8<1.2 Overall CD4:CD8>1.2 CD4:CD8<1.2 Overall

No activation TH1 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% TH0 16.7% 50.00% 25.00% 10.00% 0.00% 5.00% TH2 83.3% 50.00% 75.00% 90.00% 100.00% 95.00% Activation TH1 0.00% 40.00% 55.00% 0.00% 40.00% 55.00% TH0 66.67% 100.00% 75.00% 70.00% 0.00% 0.00% TH2 33.33% 0.00% 25.00% 30.00% 60.00% 45.00%

The level of the marker was not different between infected and non infected patients. The group without peritonitis secreted many pro- inflammatory cytokines and IL-4, which confirms data described by other groups (2,19—22) showing important immune activation in blood and peritoneal fluid of CAPD patients with and without peritonitis, with great production of pro-inflammatory and anti- inflammatory cytokines [IL-10 (22) and transforming growth factor˚β (TGFβ) (2,23)]. In the group with peritonitis, the findings of activation and secretion of cytokines were similar, but the level of IFN production was a little greater. All the CAPD patients displayed immunodeviation TH2, indicating that the patients have an immunodeficiency of the cellular type, because they are inca- pable to firing up TH1 cells. That immunodeviation was recently described by Yokohama et al (24) in asymptomatic CAPD patients, whose IFN-γ values were similar to those found in the present study. It is necessary to better define the immunodeviation TH2, owing to reports of high levels of TGFβ and IL-10 in CAPD patients. Immunosuppression and genesis of fibrosis is associated with TGFβ, as with other cytokines. Also, IL-10 and TGFβ are anti-inflammatory cytokines, and leaders in the TH3 lymphocyte subset recently described as inductors of immune tolerance.

FIGURE 2 The upper square shows TH2 cells stained with Conclusions interleukin-4 phycoerythrin (IL-4 PE) antibody and no interferon-γ Patients on CAPD without and with peritonitis showed (IFN-γ)—fluorescein isothiocyanate conjugate (FITC) in non stimu- immune activation per se, and production of pro-in- lated cells of a patient on continuous ambulatory peritoneal dialy- flammatory cytokines, accompanied by a pattern of sis with peritonitis. The lower square shows cells stained mainly γ with IL-4 and some IFN-γ after cell stimulation. Data was obtained cytokine TH2 and IFN- deficiency. Patients with peri- in a FACScalibur flow cytometer (Becton—Dickinson, Mountain tonitis secrete a little more IFN-γ and show immuno- View, CA, U.S.A.). stimulation, too. The immunostimulation tries to Kl nger et al 175 correct the immunodeviation from TH2 to TH0; how- the immune response to tuberculosis. 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