Comparative Analysis of Immunophenotypic Abnormalities in Cellular Immunity of Uremic Patients Undergoing Either Hemodialysis or Continuous Ambulatory Peritoneal Dialysis

Abstract

Aim. To investigate the abnormalities of cellular immune responses in patients on hemodialysis (HD) and in those on continuous ambulatory peritoneal dialysis (CAPD). Patients and Methods. Forty-five (45) healthy volunteers, 34 patients on HD therapy, and 37 patients on CAPD were recruited for the present study. Lymphocyte subpopulations (CD2 + , CD3 + , CD3 + /CD4 + , CD3 + /CD8 + , CD3 − /16 + 56 + , CD19, and CD4/CD8) were determined by flow cytometry. Results. Lymphopenia, decreased absolute counts, and altered percentage values of CD3 + , CD3 + /4 + , and CD19 + subpopulations were found in both patient groups. The HD and CAPD patients showed increased percentages of natural killer cells (CD3 − /16 + 56 + ) compared to controls but CD4 + /CD8 + ratio showed no significant changes among uremic patients and controls. Conclusions. Replacement therapy may contribute to the quantitative alterations of immune subsets found in HD and CAPD patients compared to normal subjects. We speculate that these changes account, at least in part, for the immune dysregulation observed in patients with chronic renal failure. Analysis of lymphocyte subsets will help the research and the evaluation of the possible causes of immunodeficiency in uremic patients undergoing replacement therapy and will probably contribute to more efficient and preventive strategies.

• cellular immunity
• lymphocyte subsets counts
• flow cytometry
• hemodialysis
• continuous ambulatory peritoneal dialysis

INTRODUCTION

Chronic renal failure is accompanied by various immunologic abnormalities of innate and acquired immunity. The clinical relevance of this altered function is related to the morbidity and mortality observed in uremic patients. The immunological deficiency due to uremia cannot be corrected by any replacement therapy, hemodialysis (HD), or continuous ambulatory peritoneal dialysis (CAPD). At the same time, infections are the main cause of hospitalization and the second leading cause of death in end-stage renal disease patients.[1] Many studies are focussing on immunologic deficiency mainly in cellular but also in humoral immunity.[1-3]

It is well established that patients with chronic renal failure exhibit peripheral blood lymphopenia, which is accompanied by a decreased delayed hypersensitivity response to a variety of antigens,[2&3] decreased lymphocyte proliferative response when stimulated by different antigens,[4&5] and decreased production of antibodies to specific stimuli by B cells.[6]

The cellular components of the immune system are identified and isolated by flow cytometry and by fluorescent staining of specific membrane antigens for each cell group. These monoclonal antibodies are called CD markers.[7]

In order to investigate the abnormalities of cellular immunity responses in uremic patients undergoing HD or CAPD, we evaluated the quantitative alterations of the uremic patients lymphocytes subset counts in comparison with those of normal subjects.

PATIENTS AND METHODS

The present study was carried out in 34 patients (16 male, 18 female) undergoing HD with mean age 63 ± 10 years and 37 patients (16 male, 21 female) undergoing CAPD with mean age 67 ± 13 years. Forty-five normal individuals (17 male, 28 female) served as the control group with mean age 36 ± 11 years. The primary causes of chronic renal failure are shown in Table 1. Lymphocyte subsets (CD2 + , CD3 + , CD3 + /4 + , CD3 + /8 + , CD19 + , CD3 − /16 + 56 + , and CD4/CD8 ratio) were determined using monoclonal antibodies (Immunotech, Coulter) and flow cytometric analysis (Table 2). Briefly, 20 μL of the appropriate monoclonal antibody was incubated with 100 μL of blood sample for 20 min in the dark. The samples were then lyzed by the ImmunoPrep reagent system (Beckman Coulter Company) and analyzed in the flow cytometer (Epics Elite ESP, Coulter). Statistical evaluation was performed by analysis of variance (ANOVA) to test differences between groups (SPSS version 10).

Table 1 Primary Causes of Renal Disease

Disease	CAPD	HD
Diabetes mellitus	9	17
Hypertension	12	6
Glomerulonephritis	7	9
Polycystic disease	2	1
Others	4	4

Table 2 Monoclonal Antibodies Used for Lymphocyte Analysis

Cluster Differentiation	Specificity
CD2 +	T lymphocytes, thymocytes
CD3 +	Mature T cells
CD3 + /4 +	T helper cells
CD3 + /8 +	T suppressor/cytotoxic cells
CD19	B lymphocytes
CD3 − /16 + 56 +	NK cells

RESULTS

The results of lymphocyte subset mean values in patients on HD and on CAPD compared to healthy subjects are presented in Table 3. In patients on HD, lymphopenia and decreased lymphocyte percentages (27.24 ± 7.37 vs. 31.79 ± 7.3, p < 0.05) were found compared to normal controls. The CD2-positive T-lymphocyte percentages were increased (82.68 ± 7.06 vs. 80.14 ± 4.6, p = NS). On the other hand, CD3 + cells (70.45 ± 8.9 vs. 73.71 ± 5.78, p = NS) and CD3 + /4 + T-helper cells (41.58 ± 9.02 vs. 45.20 ± 6.65, p < 0.05) were decreased in relation to the corresponding percentages of the control subjects. The B lymphocytes were found to be significantly lower than in healthy controls (6.80 ± 4.95 vs. 12.32 ± 3.44, p < 0.05). The HD patients showed increased percentages of natural killer (NK) cells compared to controls (16.96 ± 7.60 vs. 10.3 ± /4.10, p < 0.05) and CD4 + /CD8 + ratio showed no significant changes between HD patients and controls (1.88 ± 1.20 vs. 2.01 ± 0.72, p = NS).

Table 3 Mean Values of Phenotypic Analysis in Patients on HD, CAPD and Controls

Group	N	WBC	LYM	CD2	CD3	CD3/4	CD3/8	CD19	NK	CD4/CD8
HD	34	6641 ± 2196	27.24 ± 7.37	82.68 ± 7.06	70.45 ± 8.9	41.58 ± 9.02	26.74 ± 9.37	6.80 ± 4.95	16.96 ± 7.60	1.88 ± 1.20
CAPD	37	8377 ± 2404	22.9 ± 8.55	83.9 ± 5.5	68 ± 15.35	41 ± 7.8	24.2 ± 9.5	7.2 ± 4.35	15.22 ± 9.5	2.11 ± 1.42
CONTROL	45	7151 ± 1387	31.75 ± 7.3	80.15 ± 4.66	73.7 ± 5.8	45.2 ± 6.65	24.35 ± 6.44	12.3 ± 3.44	10.13 ± 4.1	2 ± 0.72

In CAPD patients lymphopenia was also noticed, but less evident than in HD group (CD3 + , CD3 + /4 + , CD19 + , p = NS). The NK cells were not significantly increased (15.22 ± 9.49 vs. 10.13 ± 4.10, p = NS) and the CD4 + /CD8 + ratio showed no significant changes between CAPD patients and controls (2.11 ± 1.42 vs. 2.01 ± 0.74, p = NS).

DISCUSSION

The immune system is composed of cells and molecules vigilantly defending and maintaining the homeostasis of the host. Functionally, the system consists of two branches: innate (natural, unspecific) immunity and acquired (specific) immunity. Phagocytic cells represent innate immunity. Acquired immunity has two branches: humoral immunity is represented by B cells, which secrete antibodies and cellular immunity by T cells, CD4 + and CD8 + .[8]

Cells of the innate immune system initiate the immune response by phagocytosing microorganisms and presenting antigens to T-helper cells. The CD4 cells determine the kind of specific immunity that will confront the antigenic peptide: humoral or cellular. At the same time CD8 cells are divided into cytotoxic and suppressor and Natural Killer cells (NK), although functionally belonging to the innate system, they are essential for inducing cellular immune responses.[9]

Flow cytometry is an automated method detecting blood lymphocyte subpopulations on the basis of very specific characteristics. The more extensive use of flow cytometry in immunology, hematology and oncology has lead researchers to apply it in nephrology too.[10]

Lymphopenia, decreased absolute counts, and altered percentage values of immune cells were found in the HD and in CAPD patients. This disorder was statistically significant in HD population. It was also noticed that the CD4 + /CD8 + ratio showed no significant changes among uremic patients and controls.

The decreased number of T cells that we observed is also confirmed by literature, based on the fact that uremia is characterized by lymphopenia.[11] This decline in T cells affects both CD4 + and CD8 + and that explains why CD4 + /CD8 + ratio is not significantly influenced in our study. Replacement therapy (HD or CAPD) does not affect the CD4 + /CD8 + ratio.[12&13] On the other hand, in other studies CD4 + /CD8 + ratio is increased[14] and many authors support the argument that only T helper cells are decreased in uremia.[13], [15]

Over the last decades it was well established that chronic renal failure exhibits peripheral blood lymphopenia, which is accompanied by decreased lymphocyte proliferative response when stimulated by different antigens.[11], [16] This disorder has been related to decreased production of interleukin-2 and interferon-γ and it is more apparent in HD patients.[11], [16] Uremic toxins, LDL, prostaglandin E2, and parathormone seem to be incriminated for such insufficiency.[16&17] Many theories have been established in order to explain immunological deficiencies of uremic patients. These include the direct damage of monocytes and the defect of soluble receptor of T lymphocytes in a uremic environment.[16&17] No matter what the cause of this deficiency, immunological abnormalities increase the rate of infections, affect erythropoiesis, and affect the clinical outcome of end stage uremic patients.[18]

Natural killer cells are derived from bone marrow. They play a role in the defense against infections. Their cytotoxic function is bone marrow– and cytokine-dependent.[20] The increase of NK cells that we observed (significant in HD patients) is in accordance with reports,[21&22] that found raised values of CD16 + CD56 + cells in HD and CAPD patients, along with high levels of factors that stimulate monocyte and granulocyte production.

It should be noticed that the age differences among healthy controls and uremic patients do not affect the credibility of our study. It has been known that total leukocyte and absolute lymphocyte counts are highest at birth and decline with age. Many studies relative to lymphocytes' subpopulations have shown that age does not affect them.[23-25] On the other hand, the primary cause of renal failure does not seem to affect the degree of immunodeficiency [online].

HD and CAPD appear to contribute to the state of chronic activation of the immune system resulting in a localized chronic inflammatory response. The causes and clinical consequences of this chronic activation remain unknown. These results may explain the increased vulnerability to infections in uremic patients compared with healthy subjects. We recommend lymphocyte subset determinations for detection of immune abnormalities in order to promote further research regarding mechanisms and clinical significance of lymphocyte activation.