Abstract
Background: Cryoglobulinemia is frequent in renal transplant patients. The mononuclear and polymorphonuclear neutrophil (PMN) phagocytic systems are important for the clearance of cryoglobulin immune complexes. There might be a reduced phagocytic activity in transplant patients with cryoglobulinemia (CRYO+). Methods: We studied the phagocytic activity by PMNs, in the presence of immune complexes in renal transplant patients, with or without hepatitis C virus (HCV) infection. Thirty-seven patients subjected to kidney transplant were evaluated, and for the control group, healthy blood donors were chosen. The presence of cryoprecipitate was evaluated, as well as HCV infection, phagocytic activity by neutrophils during the ingestion and digestion phase. Results: The presence of cryoprecipitate was detected in 75.7% of the patients, 39.28% of which had HCV infection. IgG, IgM, IgA, and C3 and C4 complement components were identified in the cryoprecipitate. There was a reduction in the ingestion phase of phagocytosis by PMNs in renal transplant CRYO+ though the digestion phase was preserved. Conclusion: We concluded that there was a decreased PMN activity in transplanted patients presenting cryoglobulinemia.
INTRODUCTION
Patients with chronic renal disease present a greater risk of hepatitis C virus (HCV) infection due to the constant exposure to blood-derived products and HCV infection is a frequent cause of cryoglobulinemia in renal transplant patients.Citation1–4
Sens et al.Citation5 evaluated the presence of cryoglobulinemia in patients subjected to renal transplantation, by establishing a prevalence of 74.4% with HCV infection in 37.9% of the patients with cryoglobulinemia (CRYO+).
Cryoglobulinemia is defined as the presence of immunoglobulins that precipitate with the exposure to cold and resolubilize when rewarmed, causing systemic vasculitis with the deposit of immune complexes, which are usually removed from the circulation by phagocytes.Citation6–9
After chemotaxis to injured tissues neutrophils initiate phagocytosis process that consists of adhesion, ingestion, and digestion of solid particles by cells resulting in the elimination of non-pathogenic rests. It is believed that the phagocytic system is responsible for removing immune complexes from the circulation, which makes the study of phagocytic activity relevant in relation to cryoglobulinemia.
Forte et al.Citation10 evaluated phagocytic activity in different situations, such as in mal-nourished children,Citation11 in recurring abscesses,Citation12 in patients with atopic dermatitis,Citation13 besides studying the presence of cryoglobulins in patients with systemic lupus erythematosus (SLE), and observed a reduction in phagocytosis in patients with active lupus,Citation14 concluding that the neutrophil is important for the immunity defense and immune complexes removal.
Our hypothesis was that a disturbance in polymorphonuclear neutrophil (PMN) phagocytic activity might interfere in the immune complexes clearance, leading to their deposition in different tissues.
The aim of this study is to evaluate the ingestion and digestion phase of phagocytic activity by PMN in renal transplant patients with and without cryoglobulinemia.
PATIENTS AND METHODS
Patients subjected to renal transplantation were studied, with follow-up in the Nephrology Clinic, during 11 months. The study protocol was approved by the Institution's Research Ethics Committee and informed consent was obtained from every patient. The inclusion criteria were renal transplant lasting longer than 12 months, without rejection, infections, or complications in the three preceding months or during the 15 days following material collection and stable renal function with creatinine serum levels less than 2.5 mg/dL. The immunosuppressive therapy used a triple regimen containing corticosteroid (prednisone 0.15–0.20 mg/kg/day) in combination with cyclosporine or tacrolimus and azathioprine or mycophenolate mofetil (MMF). Patients who presented clinical or laboratorial alterations were excluded by suggesting autoimmune disease, liver disease, neoplasia, infection, blood transfusion, immunization, atopy, recent surgery, primary immunodeficiency, mal-nourishment, or positive serology for hepatitis B virus (HBV) and HIV in the 3 months preceding the study as well; underlying disease and clinical data suggestive of cryoglobulinemia were also researched. For the control group, healthy, HCV-negative blood donors were chosen.
Three assays were performed to evaluate the ingestion phase by PMN: PMN incubated with zymosan (Zy), PMN incubated with Zy and homologous serum (Zy and HS), and PMN incubated with Zy and autologous serum (Zy and AS).Citation12,Citation14 A light microscope was used to evaluate the number of PMN cells that presented three or more phagocytic vacuoles within a fixed number of 200 neutrophils. All assays were performed in duplicate.
For the digestion phase phagocytosis assessment, PMNs were incubated with nitroblue tetrazolium (NBT) (spontaneous) and lipopolysaccharide (LPS) (stimulated), and the percentage of NBT reduction at a fixed number of 200 neutrophils was determined. All assays were performed in duplicate.
For the evaluation of the digestion phase by PMNs, NBT was used, which is a yellow and soluble dye. The NBT reduces into dark blue particles, called formazan, that are deposited in the neutrophil cytoplasm. The NBT test becomes more evident when stimulated by substances such as LPSs. This test can, therefore, evaluate the release of electrons that occurs in the digestion phase by oxidative metabolism of the PMNs.Citation10
For the cryoprecipitate analysis, peripheral blood samples were collected with their serums stored at 4°C for 15 days. The cryoprecipitate components (IgG, IgM, IgA, C3, and C4) were evaluated by using simple radial immunodiffusion. HCV detection in serum was done by using the polymerase chain reaction (PCR).
The data were presented as means and standard deviations in the statistical analysis. Comparisons among the different groups were made by using the Mann–Whitney test with a significance level of 5% (p < 0.05).
RESULTS
Thirty-seven patients, 21 male and 16 female, subjected to kidney transplantation were selected between 19 and 70 years old (mean: 39.51 ± 12.29 years). The control group was composed of 37 healthy individuals, being 11 male and 26 female, ranging from 14 to 69 years (mean: 23.35 ± 12.52 years). The presence of a cryoprecipitate was detected in 75.5% of the patients. G, M, and A immunoglobulins were identified in the cryoprecipitate, as well as C3 and C4 complement components. Among the patients with cryoprecipitate formation, 39.28% had HCV infection detected by PCR in serum. The etiology was not determined in the remaining patients.
shows the means of the PMN phagocytosis with Zy particle ingestion (control), with Zy and HS, and with Zy and AS of CRYO+, patients without cryoglobulinemia (CRYO−), and healthy groups. The percentage of PMN that presented three or more phagocytic vacuoles within a fixed number of 200 PMN cells was evaluated. A significant difference was established between the CRYO+ and the CRYO− groups (p < 0.05) and also between the CRYO+ group and the healthy individual (p < 0.05). There was no relevant difference between the CRYO− group and the healthy individual.
TABLE 1. Values of the arithmetic means and standard deviations of phagocytosis ingestion phase by neutrophils of renal transplant patients with cryoglobulinemia, without cryoglobulinemia, and healthy individuals: percentages of cells that presented three or more phagocytic vacuoles within a fixed number of 200 cells
Regarding the NBT assay, demonstrates the arithmetic means of the spontaneous and stimulated NBT test in the CRYO+, CRYO−, and healthy groups. All comparisons did not statistically demonstrate significant differences referring to phagocytosis digestion phase.
TABLE 2. Values of the arithmetic means and standard deviations of the NBT test in renal transplant patients with cryoglobulinemia, without cryoglobulinemia, and healthy individuals
Regarding the immunosuppressive therapy, shows the mean doses of immunosuppressive drugs and comparison between CRYO+ and CRYO– renal transplant patients. The comparison of the mean doses of cyclosporine, tacrolimus, azathioprine, and MMF was not statistically significant between CRYO+ and CRYO– renal transplant patients (p>0.05) except with azathioprine with a higher dose in CRYO– patients (p < 0.05).
TABLE 3. Values of the arithmetic means and standard deviations of the mean doses of immunosuppressive therapy in CRYO+ and CRYO− renal transplant patients
DISCUSSION
The analysis of the phagocytosis ingestion phase by PMNs showed a significant reduction when the cells were incubated with HS and when the cells were incubated with AS in CRYO+ transplanted patients when compared to healthy individuals. The results showed no difference between the two assays when compared to healthy individuals. The fact that no difference was evidenced between the assays with HS and AS suggest that the reduction is probably due to an intrinsic problem of the PMNs. Parallel studies demonstrated normal serum levels of C3 complement component. This data exclude the possibility that the reduction of phagocytosis was a problem related to C3 component. These data suggest that the reduction is more likely to be resulting from a cellular intrinsic problem.Citation15–17
The results of phagocytosis ingestion phase by PMNs showed no difference among the CRYO− transplanted patients and healthy individuals, suggesting a normal PMN phagocytosis ingestion phase in these groups. It is possible that the PMN can lead to a clearance of the circulating immune complexes in CRYO+ patients. Furthermore, the deficiencies in PMN function can increase the risk of infection. These deficiencies can be primary, secondary to other pathologies, or in association with other genetic alterations.Citation15
The use of serum in the phagocytosis technique induces complement activation by Zy, which results in the activation of C3b and C5b that bind to these particles, with opsonization and consequent ingestion. The assay which uses only Zy and PMN, considered control, evaluates spontaneous cell ingestion, without phagocytic stimuli, in addition to evaluating the method's viability.Citation14 PMNs are involved in the pathogenesis of several inflammatory diseases; one mechanism that generates neutrophilic inflammation is the deposit of immune complexes in the tissues.Citation18 Lazzarin et al.Citation19 studied patients with hepatitis A, B, and acute non-A and non-B hepatitis: circulating immune complexes were found in the serum of these patients with inhibitory effect upon the phagocytic activity of neutrophils. The Fcγ receptors are important for phagocytic activity, and Hundt et al.Citation20 demonstrated that, besides the cytokines, immune complexes can induce the expression of FcγRI and significantly reduced FcγRII and FcγRIII expression in the synovial fluid of rheumatoid arthritis patients.
In this study, during the evaluation of the phagocytosis digestion phase by PMNs through the NBT test, a significant difference was not demonstrated among the different patients groups analyzed.
The PMN activity needs to be whole preserved to make the immune complexes clearance. There are immune deficiencies that only affect phagocytosis ingestion phase. Cytokines as IL-12 and γ-interferon could interfere in its activity.
In addition, in this study a high prevalence of cryoglobulinemia in renal transplant patients was observed, with the cryoprecipitate constituted of G, M, A immunoglobulins, and C3 and C4 complement components; HCV infection was found in CRYO+ patients.
The prevalence of cryoglobulinemia in this study was very high (75.5%) compared to previous reported prevalence. Cryoglobulins were isolated from serum samples after they had been stored for 15 days at 4°C, period of longer storage compared to other studies. We realized that the formation of cryoprecipitate in some cases occurred only after 7 days of its storage. The longer period of observation may explain our high prevalence of cryoglobulinemia.
Several studies have revealed the association of cryoglobulinemia with HCV infection through anti-HCV antibody and HCV-RNA (ribonucleic acid) analysis.Citation21–26 Clinical manifestations which can possibly be associated with the presence of cryoglobulinemia among renal transplant patients, were not frequent in this and other studies. Similarly in one study concerning patients who were CRYO+ associated with chronic HCV infection, only 13.1% presented signs or symptoms of cryoglobulinemia.Citation27 Hemodialysis and immunosuppression associated with kidney transplantation may be important factors benefiting the cryoglobulinemia clinical manifestations suppression in patients presenting subclinical disease.Citation28 Among renal transplant patients with arthralgia, purpura, proteinuria, or peripheral neuropathy of undetermined etiology we must think about cryoglobulinemia.Citation5
The role of immunosuppression in affecting neutrophilic activity cannot be ruled out although there was no significant difference between CRYO+ and CRYO− groups with cyclosporine, tacrolimus, and MMF, but when azathioprine was analyzed there was a significant difference with a higher dose in CRYO− patients.
We concluded that there was a reduction in the ingestion phase of phagocytosis by PMN in CRYO+ renal transplant patients and the digestion phase was conserved both in CRYO+ and CRYO− patients. Thus, we believe that the reduction of PMN activity in CRYO+ renal transplant patients might have interfered in the clearance of immune complexes.
ACKNOWLEDGMENTS
Financial support for this study was provided by CAPES – Coordination for the Improvement of Higher Level – or Education – Personnel.
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