Lymphocytes Subsets in the Course of Continuous Ambulatory Peritoneal Dialysis (CAPD)

Abstract

Background: We studied lymphocyte subset counts in comparison with normal subjects in order to clarify the abnormalities of cellular immune responses in uremic patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Methods: The study included 37 CAPD patients and 45 normal individuals, as the control group. For the study, CAPD patients were divided into four groups depending on duration of replacement therapy. Group I consisted of patients treated for 0–6 months (n = 6), group II for 6–12 months (n = 6), group III for 13–24 months (n = 16), and group IV for more than 25 months (n = 9). Flow cytometry was used for estimation of lymphocyte subsets (determination of CD2, CD3, CD3 +/CD4 +, CD3 +/CD8 +, CD3 −/16 + 56 +, CD19, CD4/CD8). Results: Our patients started CAPD with decreased lymphocyte subset counts, slightly above the normal range (excluding CD3 −/16 + 56 +, CD2). After 6 months of CAPD therapy, an increase in CD4/CD8 ratio was observed and all examined lymphocyte subset counts decreased (excluding CD2). In patients on CAPD for more than 25 months, CD3 +/CD4 +, CD19 counts were below the normal range, CD3 −/16 + 56 + exceeded the upper limit of normal range and at the same time mean total lymphocyte count (TLC) was maintained in the normal range. Conclusions: We recommend lymphocyte subset determinations for detection of immune abnormalities in the course of CAPD treatment.

• Lymphocytes subsets
• Flow cytometry
• CAPD

Introduction

One choice of replacement therapy in patients with end‐stage renal disease is continuous ambulatory peritoneal dialysis (CAPD), where most serious complications are peritonitis and fibrosis, resulting from failure of the dialysis technique.[1] When germs enter the peritoneum, cells of the immune system act in defense and trigger tissue injury. Functionally, the system has two branches: natural (innate, unspecific) immunity and specific (acquired) immunity.[2] Acquired immunity consists of two branches: humoral immunity managed by B cells, which secrete antibodies or immunoglobulins, and cellular immunity managed by T cells, CD4 +, and CD8 +.[3], [4] Those components are identified and isolated by flow cytometry, the staining of proteins specific for each cell group with fluorescent monoclonal antibodies called CD markers, the role of which in host immunity is just beginning to be understood.[3]

It is well established that chronic renal failure exhibits peripheral blood lymphopenia, which is accompanied by a decreased delayed hypersensitivity response to a variety of antigens,[4], [5] decreased lymphocyte proliferative response, when stimulated by different antigens,[6], [7] and decreased production of immunoglobulins by B cells to specific stimuli.[8] The clinical relevance of altered lymphocyte function is not well understood. At the same time, infections are the second leading cause of death in hemodialysis patients[9] and peritonitis is the primary complication in CAPD patients.[10], [11] Although numerous investigations of lymphocytes from hemodialysis patients have been reported, there are few published studies of peritoneal lymphocytes in CAPD patients.

We studied, as immunological parameters, lymphocyte subset counts in comparison with normal subjects in order to clarify the abnormalities of cellular immune responses in uremic patients undergoing CAPD and to evaluate whether estimation of lymphocyte subset counts can be more helpful than total lymphocyte count (TLC) in earlier diagnosis of immune changes in the course of CAPD.

Patients and Methods

The study included 37 CAPD patients (21 female, 16 male, age: 66,88 ± 13,48 M ± SD) and 45 normal individuals (28 female, 17 male, age: 35,8 ± 10,8 M ± SD) who served as our control group. Primary causes of chronic renal failure were diabetes mellitus (17), hypertensive nephropathy (6), glomerulonephritis (9), polycystic kidney disease (1), others (4). The study population was divided into four groups depending on duration of replacement therapy. Group I consisted of patients treated for 0–6 months, group II for 6–12 months, group III for 13–24 months, and group IV for more than 25 months.

Lymphocyte subsets (CD2 +, CD3 +, CD3 +/4 +, CD3 +/8 +, CD19 +, CD3 −/16 + 56 +, CD4/CD8 ratio) were quantitated using monoclonal antibodies (Immunotech, Coulter) and flow cytometric analysis (Table 1). Briefly, 20 µL of the appropriate monoclonal antibody was incubated with 100 µL of a blood sample for 20 min in the dark. The samples were then lysed by ImmunoPrep reagent system (Beckman Coulter Company) and analyzed in the flow cytometer (Epics Elite ESP, Coulter).

Table 1. 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

Student's t‐test was performed to test differences between groups (SPSS vs. 10).

Results

Table 2 shows immunophenotypes of patients on CAPD. CAPD patients showed increased natural killer cells than controls (15.22 ± 9.49 vs. 10.13 ± 4.10, p = NS). Mean CD4/CD8 ratio levels were slightly higher in CAPD patients compared with controls (2.11 ± 1.42 vs. 2.01 ± 0.74, p = NS). CAPD patients showed lower lymphocyte subpopulations comparing with controls and especially CD3, CD3 +/4 +, CD19 + were lower than healthy subjects (p = NS).

Table 2. Immunophenotypes of Patients on CAPD

Groups	N	(WBC)	(LYM)	(CD2)	(CD3)	(CD3/4)	(CD3/8)	(CD19)	NK	NK like	CD4/CD8
1	6	8995 ± 2478	22.68 ± 4.99	81.5 ± 4.5	72.75 ± 7.33	40.8 ± 6.4	29.2 ± 8.6	9.95 ± 4.55	11.75 ± 6.1	2.7 ± 1.8	1.56 ± 0.75
2	6	6950 ± 1427	21.6 ± 6.13	84.15 ± 7.7	68.8 ± 15	38.7 ± 4.5	26 ± 11.8	6.3 ± 4.16	15.4 ± 8.4	1.9 ± 1.33	1.7 ± 0.6
3	16	8214 ± 2625	24.1 ± 11.24	84.12 ± 5.4	65.6 ± 19.9	43.2 ± 9.9	21.7 ± 9.3	6.9 ± 4.3	16.7 ± 11.3	2.9 ± 2.14	2.46 ± 1.6
4	9	9208 ± 2309	21.77 ± 6.88	84.97 ± 4.96	70.6 ± 9.9	39 ± 5.8	24 ± 8.9	6.43 ± 4.47	14.8 ± 9.13	2.7 ± 1.9	2.15 ± 1.42
Total	37	8377 ± 2404	22.9 ± 8.55	83.9 ± 5.5	68.0 ± 15.35	41 ± 7.8	24.2 ± 9.5	7.2 ± 4.35	15.22 ± 9.5	2.7 ± 1.9	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	3.14 ± 2.54	2.0 ± 0.72

Our uremic patients started CAPD with decreased lymphocyte subset counts slightly above the normal range (excluding CD3 −/16 + 56 +, CD2). After 6–12 months of CAPD therapy, a small increase in CD4/CD8 ratio was observed (p = NS) and all examined lymphocyte subsets counts decreased (excluding CD2) (p = NS). In the study period of 13–24 months, all examined lymphocyte subset counts slightly increased (excluding CD3, CD3/8) (P = NS). In patients on CAPD for more than 25 months, CD3 −/16 + 56 exceeded the upper normal limit of normal range (p = NS) and at the same time mean TLC was maintained in the normal range.

In general, we could not find any significant difference in lymphocyte subsets in the course of CAPD, probably because of our small study population.

Discussion

The immune system is composed of cells and molecules vigilantly defending and maintaining the homeostasis of the host. Functionally, the system has two branches: natural (innate, unspecific) immunity and specific (acquired) immunity. Phagocytic cells manage innate immunity. Acquired immunity has two branches: humoral immunity managed by B cells, which secrete antibodies or immunoglobulins, and cellular immunity managed by T cells, CD4 +, and CD8 +.[4]

Cells of the innate immune system (neutrophils, monocytes, eosinophils, and dendritic cells) start and amplify the immune response by phagocytosis of germs and antigens, presenting them to T helper cells from the specific immunity system. The T cells determine the kind of specific immunity that will fight the antigen: humoral or cellular.[2] The CD8 cells are divided into cytotoxics and suppressors and the natural killer cells (NK) are functionally from the innate system, but they are essential for inducing cellular immune responses.[2]

Specific cellular immunity, which is mediated by T cells, and defects offering these lymphocytes underlie the most severe immune deficiencies. Because antibody production requires intact T cell function, most T cell defects lead to combined (cellular and humoral) immune deficiency.

Lymphocytes normally comprise 20–40% of peripheral blood leukocytes. Of these, about 70–80% are T lymphocytes and 10–20% are B lymphocytes. T cells can further be classified into cytotoxic T, helper T, and suppressor T cells. Helper T and suppressor T cells serve as immunoregulatory cells of the immune response. Altered numbers of immune cells contribute to immunologic abnormalities, depressed erythropoiesis, increased infection rates,[12] and poor outcome.[13] Early detection of immunologic disturbances may initiate early clinical intervention, resulting in more effective treatment with peritoneal dialysis. Repeated determinations of T lymphocyte counts seem to be helpful in the early diagnosis of such disturbances.

When comparing the percentages of peripheral blood T cells from CAPD patients with normal controls, we found no significant differences. The CD4/CD8 ratio levels were higher in CAPD patients compared with controls (p = NS). Other researchers[14], [15] have verified these findings. On the other hand, an increase in the percentage of activated T cells in the peripheral blood of CAPD patients as compared to normal controls have been reported by other authors.[16] The reasons for this considerable variability are unknown. As has been referred, the proportion of CDs subset counts varies with age.[17] In CAPD patients, it is possible that the decrease in these lymphocyte subsets has a role in the decreased secretion of IL‐2 by lymphocytes found in these patients.[18]

Natural killer cells are derived from bone marrow. They play a role in the defense against infection.[19] Their cytotoxic function is bone marrow and cytokine dependent.[20] The expansion of NK that we observed, although not significant, accords with reports,[21], [22] which found expansion of CD14 + CD16 + cells in CAPD patients, along with high levels of factors that stimulate monocyte and granulocyte production.

In the current study, we found no significant changes in lymphocyte subsets in the course of CAPD. Shohat et al.[14] found no changes in the percentage or absolute number of peripheral T cells after 6–9 months of CAPD therapy.[14] In spite of this finding, repeat determinations of CD3, CDE +/CD4 +, CD3 +/CD8 +, and CD19 + cell counts indicate that these counts decrease earlier than an evaluation of TLC indicates. This observation is confirmed by the recent literature.[15] The significance of these findings in the patients' lymphocytes suggests an activated and proinflammatory environment.

In the first months of CAPD therapy, we saw an improvement in immune status as expressed by an increase in CD4/CD8 ratio. It is referred that CAPD patients with lower ratio have more lymphopenia, more CD8 cells, especially the suppressor type. Those findings are controversial and a need for more research and study about the CD4/CD8 ratio in clinical CAPD settings such as transplantation is needed.[16]

Natural killer cells are derived from bone marrow. They play a role in the defense against infection.[17] Their cytotoxic function is bone marrow and cytokine dependent.[18] In our patients, we observed an increase in the number of natural killer cells over the normal range with length of time n CAPD. That finding is in accordance with the study performed on CAPD patients by Palop et al.[19] Natural killer cells and their precursors are more resistant to immunosuppressive agents than are other immunocompetent cells, so that suppressive uremic toxins affect the number and function of T lymphocytes more effectively than they affect the number and function of natural killer cells. In patients on CAPD, episodes of peritonitis and exit‐site and tunnel infection may stimulate production of natural killer cells.[20]

It should be noted that age difference 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 lymphocyte subpopulations have shown that age does not affect them.[21], [22] On the other hand, the primary cause of renal failure does not seem to affect the degree of immunodeficiency.[23]

Peritoneal dialysis appears to contribute to the state of chronic activation of the immune system, resulting in a localized chronic inflammatory response. Although the majority of our results are not significant, because of the small study population, they remain obvious and guide us to further investigation. The causes and clinical consequences of this chronic activation remain unknown. In spite of this, we recommend lymphocyte subset determinations for detection of immune abnormalities in the course of CAPD treatment because the activated state of these cells and their secreted cytokines may contribute to long‐term pathology of the peritoneum, such as progressive fibrosis and loss of ultrafiltration. The possibility of such an influence should be taken into account and further research is needed to determine mechanisms and clinical significance of lymphocyte activation.