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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Patients with chronic kidney disease (CKD) display a high prevalence of cardiovascular events and acute infections. Potential effector cells are the CD16+ monocytes, known to be increased in the peripheral circulation in CKD. The aim of this study was to assess the expression of CD16 and CX3CR1 on peripheral and in vivo extravasated monocytes in patients with CKD (GFR < 20 ml/min × 1.73 m²) using flow cytometry. In vivo extravasated monocytes were collected from a local inflammatory site, induced by a skin blistering technique. Soluble markers were assessed by Luminex. The number of CD16+ monocytes was significantly higher in patients with CKD compared with healthy subjects, both in the peripheral circulation (P < 0.05) and at the site of induced inflammation (P < 0.001). Patients with CKD displayed significantly higher concentration of soluble CX3CL1 both in the peripheral circulation (P < 0.01) and in the interstitial fluid (P < 0.001). In addition, patients with CKD had a significantly higher concentration of TNF-α in the peripheral circulation (P < 0.001). On the contrary, at the inflammatory site, concentrations of both TNF-α and IL-10 were significantly lower in patients with CKD compared with healthy controls (P < 0.05 for both). In conclusion, patients with CKD have an increased percentage of CD16+ monocytes in both circulation and at the inflammatory site, and this finding is in concurrence with simultaneous changes in CX3CR1. Together with distorted TNF-α and IL-10 levels, this may have potential impact on the altered inflammatory response in CKD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Morbidity and mortality remain high in patients with chronic kidney disease (CKD), predominantly due to cardiovascular and infectious diseases. Cardiovascular mortality is up to 500-fold higher in patients with CKD, compared with age-matched subjects displaying normal renal function [1]. This is partly explained by acceleration of atherosclerosis due to impaired endothelial function and low-grade inflammation, both associated with incipient and progressive CKD. Disturbances in bone mineral metabolism have also been recognized as key factors contributing to increased risk of morbidity and mortality in CKD [2]. In addition, acute infections contribute substantially to the high rates of hospitalization and mortality. Data suggest that the annual mortality rate for pneumonia and sepsis is up to 100-fold higher in patients with CKD compared with the general population [3-5].

Monocytes play a crucial role both in atherogenesis and in host response to infection. In early atherosclerosis and interstitial inflammation, monocytes migrate into the arterial intima and subsequently into the subendothelial space. When exposed to immune modulators, such as tumour necrosis factor-α (TNF-α), IL-10, monocyte chemotactic protein-1 (MCP-1, CCL2) and macrophage inflammatory protein-1α (MIP-1α , CCL3), monocytes alter their expression of adhesion molecules promoting extravasation and subsequent cellular activation. Whereas TNF-α merely reflects the proinflammatory reaction, IL-10 is associated with the anti-inflammatory response [6-8].

The existence of monocyte subpopulations in peripheral blood is well established. Flow cytometry can, in addition to identifying the monocyte characteristic CD14+ surface marker, distinguish a CD16+ subpopulation known to expand in states of inflammation. It represents less than 10% of the total peripheral monocyte population in healthy controls and is increased in peripheral blood in numerous inflammatory conditions, including patients with CKD [9-15].

Moreover, it is possible to split the CD16+ monocytes into two subgroups referred to as intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes, respectively. When intermediate and non-classical monocytes are not separated, they should be addressed to as CD16+ monocytes only [16].

Expression of the chemokine receptor CX3CR1 is crucial for transmigration of the CD16+ phenotype [17], but it is not known whether CD16+ monocytes are in fact enriched at the actual site of inflammation, for example, in the interstitium [18].

We have, in a number of previous studies, using a well-established skin chamber technique, assessed the functional response of in vivo extravasated leucocytes. These studies demonstrate an altered leucocyte response at the actual site of inflammation in patients with CKD and in atherosclerosis [19-26]. Given an important immune-regulatory role of CD16+ monocytes, we addressed the question whether an increased number of peripheral cells is paralleled by an analogous increase in this subpopulation at an inflammatory site. In vivo extravasated monocytes were therefor collected by the skin chamber model and studied by flow cytometry. In addition, IL-10, TNF-α and CX3CL1, which are important mediators for migration and activation of the CD16+ monocytes, were assessed by immune assays.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Patients and healthy controls

The study enrolled 12 patients with CKD (eight males and four females) with a mean age of 61 ± 6.2 years. GFR was calculated according to the modification of diet in renal disease formula [27] and was below 20 ml/min × 1.73 m2, and the patients were not yet on dialysis. The aetiology of renal impairment varied from glomerulonephritis, interstitial nephritis, adult polycystic kidney disease, amyloidosis and nephrosclerosis. None of the patients suffered from heart failure. Patients with known active systemic inflammatory disease, infectious disease, diabetes mellitus as well as those prescribed antibiotics, corticosteroids, non-steroid anti-inflammatory drugs, statins, warfarin or immunosuppressive agents were excluded. Mean body mass index (BMI) was 26 ± 3.8 kg/m2. One of the 12 patients was a smoker, and five patients had a family history of kidney disease. Current medication of the patients with CKD is displayed in Table 3. In addition, 12 age- and gender-matched healthy subjects were included as a control group. Mean age was 60 (±7.8) years, and none had any clinical signs of disease or laboratory findings indicating kidney disease. The mean BMI in the healthy subjects was 25 ± 3.5 kg/m2, and one of the 12 was an active smoker.

Patients with CKD were recruited from the department of nephrology at Karolinska University Hospital. All participants gave informed written consent, and the study was approved by the local ethical committee at the Karolinska University Hospital, Stockholm, Sweden.

Preparation of peripheral leucocytes

Blood was drawn in Vacutainer tubes containing 0.129 M Na citrate (Becton Dickinson, Plymouth, UK) and kept on ice to prevent complement activation and modulation of adhesion molecule expression.

The erythrocytes were haemolysed by addition of 2 ml isotonic solution (154 mm NH4Cl, 10 mm KHCO3 and 0.1 mm EDTA, pH 7.2) per 100 μl of blood and incubated at 4 °C for 5 min. This was followed by centrifugation at 300 g, 4 °C and a wash with phosphate buffer saline (PBS), pH 7.4, before assessment by flow cytometry.

Collection of in vivo extravasated leucocytes, the skin chamber method

This method has previously been described in detail [19-26]. Briefly, a cutaneous inflammation was induced by a skin blistering technique. Two blisters, 9 mm in diameter, were raised on the volar surface of the forearm by a constant vacuum of 300 mmHg and gentle heating for 2–3 h. The following morning (after 12–14 h) the blister roofs were carefully removed and sterilized in open-bottom plastic skin chambers were mounted over the exposed blister floor and filled with autologous serum, with the addition of heparin. The autologous serum was collected the day before, centrifuged and immediately frozen at −70 °C. After 10 h of incubation, the chamber fluids were aspirated and the chambers were washed with equal volumes of PBS, which was added to the collected fluids, and the samples were placed on ice. The time of incubation is chosen based on our previous kinetic experiments showing an optimal number of accumulated cells after 10 h.

The chamber exudates were centrifuged at 300 g for 5 min at 4 °C, and cell-free chamber supernatants were stored at −70 °C and used later to measure soluble factors and chamber leucocytes, were resuspended in PBS and kept on ice until further examination.

Cell count in the chambers and in the peripheral circulation

The leucocyte populations were counted using a flow cytometer (Epics Elite, Beckman Coulter Inc, Hialeah, FL, USA), and the percentage of neutrophils, monocytes and lymphocytes were calculated. Cell count in peripheral blood was calculated on 100 μl haemolysed, stabilized and fixed peripheral blood according to the Multi-Q-Prep ImmunoPrep technique (Beckman Coulter Inc).

Immunostaining for surface markers and flow cytometry

Leucocytes from blood and the chambers were resuspended in 100 μl PBS per tube and labelled with antibody or isotype control (Table 1) for 30 min on ice. After washing with PBS, the cells were resuspended in 300 μl PBS, and the monocyte population was selected and analysed for surface markers by flow cytometry (Epics Elite, Beckman Coulter Inc). The results are expressed as mean fluorescence intensity (MFI) or as the percentage of positive cells and MFI of the positive cells. The percentage of positive cells was determined by the respective isotype control. Intermediate and non-classical monocytes were not separately defined; hence, the studied subpopulation is addressed to as CD16+ monocytes exclusively. Gating procedure for the CD16+ population is viewed in Figure 1, and gating of CX3CR1 has previously been described [28].

Table 1. Utilized antibodies against surface markers assessed by flow cytometry
AntibodyCloneConjugationCompanyμl antibody/100 μl
CD14M5E2PEBD Biosciences (San Jose, CA, USA)15
CD16NKP15FITCBD Biosciences15
CX3CR12A9-1FITCMedical and Biological Laboratories Co., Ltd (Naka-ku Nagoya, Japan)20
IgG1MOPC-21PEBD Biosciences15
IgG2b27–35FITCBD Biosciences20
image

Figure 1. CD16+ expression viewed as flow cytometric charts. Figure illustrating representative pictures of samples from the peripheral circulation and the skin chamber in patients with chronic kidney disease and healthy controls, respectively.

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Determination of soluble inflammatory mediators in chamber fluid and in serum

Soluble inflammatory mediators in serum and chamber fluid were assessed by Milliplex 26-plex (Millipore Corp, St. Charles, Missouri, USA) according to the provided manufacturers' protocol. Chamber fluids were diluted 1:4 before assessment; which is corrected for in the final results. The limit for cytokine detection using this method was 12 pg/ml.

CX3CL1 was assessed by a commercial ELISA (Quantikine immunoassay, R&D systems, Abingdon, UK), and the chamber fluid was diluted 1:3, which is corrected for in the final results.

Statistical analysis

Results are expressed as mean ± standard deviation for the normally distributed data: age, BMI, serum creatinine, eGFR, C-reactive protein (CRP), haemoglobin, PTH, phosphorus and albumin. Nonparametric data; cell count, CD16+, CX3CR1 and soluble factors are presented as median and 25–75% interquartile range. Box plots represent 25–75% interquartile range with a line at the median and bars at the non-outlier values. Statistical analysis and comparison between groups were performed using Mann–Whitney U-test. Statistical significance was determined at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Baseline characteristics of patients and healthy controls

Patients had a mean serum creatinine of 438 ± 135 μm and a mean eGFR of 12 ± 3.2 ml/min × 1.73 m2 calculated according to the MDRD formula [27]. In addition, mean values of patients were as follows: haemoglobin 120 ± 10 g/l, total calcium 2.3 ± 0.2 mm; phosphorus 1.5 ± 0.3 mm; albumin 33 ± 3 g/l; intact PTH 269 ± 91 pg/ml; and CRP 6.1 ± 2.8 mg/l. In healthy subjects, mean serum creatinine was 84 ± 14.8 μm and mean CRP was 1.9 ± 2.0 mg/l. Pharmacological treatment were shown in Table 2.

Table 2. List of prescribed medication in the patients with CKD
MedicationNumber of patients (n = 12)
Angiotensin-converting enzyme inhibitor5
Angiotensin receptor blocker6
Diuretics9
Beta-blocker4
Calcium antagonist7
Erythropoiesis-stimulating agents8
Iron supplement (iv)2
Iron supplement (oral)2

Number of leucocytes in the peripheral circulation and following extravasation

In peripheral blood, the number of leucocytes was 5374 (4004-6809) cells/μl in patients and 4999 (3476-6009) cells/μl in healthy controls with 5.7 (4.6–9.3)% and 8.6 (7.0–11.3)% monocytes, respectively, P = NS between patients and healthy controls. Median cell count in the skin chamber was 2380 (970-4353) ×103 and 1995 (1115–2940) ×103 in patients and healthy controls, respectively, P = NS. The proportion of monocytes in the skin chamber was 7.5 (7.2–14.5)% in patients and 12.5 (9.2–15.7)% in healthy subjects, P = NS.

Percentage of CD16+ monocytes in the peripheral circulation and following in vivo extravasation

The percentage of monocytes with the CD16+ phenotype in the peripheral circulation was significantly higher in patients with CKD, 22.1 (15.2–28.8)%, compared with healthy controls, 13.3 (8.9–14.1)%, P < 0.05 (Figure 2). Following in vivo extravasation in the skin chamber, the percentage of CD16+ monocytes increased to 80.2 (74.5–83.8)% in patients and to 43.0 (30.1–56.0)% in healthy controls, P < 0.001 (Figure 2). The CD16 monocyte percentage was 11.5 (7.8–16.9)% in patients and 32.9 (20.6–52.8)% in healthy controls in the skin chamber fluid.

image

Figure 2. The percentage of CD16+ monocytes assessed by flow cytometry. Patients with chronic kidney disease had significantly higher percentages of CD16+ monocytes in the peripheral circulation as well as following extravasation, P < 0.05 and P < 0.001, respectively, compared with healthy controls. Boxes represent 25–75% interquartile range with median indicated by a line.

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Expression of CX3CR1 and concentration of CX3CL1 levels in the peripheral circulation and at the site of interstitial inflammation

The majority of monocytes in the peripheral circulation expressed CX3CR1, both in patients with CKD and in healthy controls (Table 3). Following extravasation, the expression of CX3CR1 decreased by 30–40% in both patients and healthy controls (Table 3). Patients with CKD had significantly higher levels of soluble CX3CL1, both in the peripheral circulation (P < 0.01) and at the site of interstitial inflammation (P < 0.001), as compared to the healthy controls (Table 3).

Table 3. Expression of cell surface markers on monocytes from the peripheral circulation and following extravasation to the skin chamber, assessed by flow cytometry
 Peripheral circulationSkin chamberSignificance patients versus controls
PatientsControlsPatientsControlsBloodChamber
  1. Values represent median and 25–75% interquartile range, n = 12. Mann–Whitney U-test was assessed for comparison between patients and controls.

CX3CR1 (%)93.7 (92.2–95.2)95.8 (94.8–96.5)66.0 (46.9–71.9)57.9 (38.3–65.9)<0.05NS
CX3CR1 (MFI)6.1 (4.8–6.9)5.6 (5.0–6.2)2.8 (2.4–3.5)2.9 (2.7–3.6)NSNS

Soluble inflammatory markers

Patients with CKD had a significantly higher concentration of TNF-α in the peripheral circulation compared with healthy controls, P < 0.001 (Table 4). On the contrary, in the skin chamber fluid, the concentrations of both TNF-α and IL-10 were significantly lower in patients with CKD compared with healthy subjects, P < 0.05 for both (Table 4). MCP-1 concentration in peripheral circulation was similar in patients and healthy controls (NS), whereas levels in blister fluid were not detectable (ND). No significant differences were obtained between patients and healthy controls in MIP-1α levels (NS) in blood or skin chamber fluid.

Table 4. Concentration of soluble factors in the peripheral circulation and at the site of interstitial inflammation (skin chamber), assessed by Luminex and ELISA
 Peripheral circulationSkin chamberSignificance patients versus controls
PatientsControlsPatientsControlsBloodChamber
  1. Values represent median and 25–75% interquartile range, n = 12. Mann–Whitney U-test was assessed for comparison between patients and controls.

TNF-α (pg/ml)88 (55–112)32 (20–36)83 (57–146)150 (107–919)<0.001<0.05
IL-10 (pg/ml)16 (16–28)12 (12–24)12 (12–32)98 (12–119)NS<0.05
CX3CL1 (ng/ml)1.2 (0.9–1.5)0.6 (0.4–0.8)0.6 (0.4–0.7)0.3 (0.2–0.3)<0.01<0.001

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

We demonstrate for the first time that patients with CKD have an increased percentage of CD16+ monocytes both in the peripheral circulation and at sites of induced interstitial inflammation. The ratio of the percentage of CD16+ monocytes between the peripheral circulation and the interstitial inflammatory site was comparable, indicating similar transmigration capacity of the CD16+ monocytes in patients with CKD and healthy controls resulting in a comparable transmigration rate. We therefore suggest that the higher percentage of cells at the interstitial inflammation in patients with CKD is a consequence of the increased peripheral CD16+ pool.

An increased percentage of peripheral circulating CD16+ monocytes have previously been reported in patients with CKD [11-15], which is in accordance with the present data. The aim of this study was to address the question whether this fact also results in a pronounced accumulation at the actual site of interstitial inflammation. For this purpose, we applied the skin blister model to assess CD16+ monocytes following in vivo extravasation to an induced site of inflammation. During the past decade, this method has been used to evaluate leucocyte functions following in vivo extravasation in healthy controls as well as in patients with allergy, coronary artery disease and renal disease [19-26, 28, 29].

The percentage of CD16+ monocytes was almost twice as high following extravasation to the interstitial site in patients with CKD, as compared to that in healthy controls. However, the ratio between the skin chamber and the circulation was similar in both groups. This indicates that there is no increased transmigration capacity explaining the pronounced accumulation of CD16+ monocytes in patients with CKD. Furthermore, this finding is in agreement with a previous report from our group assessing the number of CD16+ monocytes in patients with coronary artery disease and normal renal function [26]. However, we cannot totally rule out the influence of local differentiation on the number of CD16+ monocytes at a given time point. Interestedly, a recent study describes a simplified skin chamber technique that generates more information on cell kinetics in an undisturbed milieu [30]. However, due to the relative low cell numbers obtained, the possibility to collect analysable RNA/DNA is challenged [31].

Fractalkine, CX3CL1, is a membrane-anchored chemokine that is released from the cell surface by proteolysis. It binds CX3CR1, and by these dual forms, fractalkine functions as a chemokine as well as an adhesion molecule for a wide variety of immune-regulatory cells [32, 33]. The CD16+ monocytes have a high expression of CX3CR1, which enhances accumulation of these cells at sites of overexpressed CX3CL1 [34]. In the present study, the expression of CX3CR1 was above 90% on monocytes in the peripheral circulation in both groups, and the expression decreased substantially following extravasation in both patients and healthy subjects, probably due to shedding. Patients with CKD had a significantly higher concentration of CX3CL1 in both blood and the interstitial fluid, which is in accordance with our data on CD16+ monocyte counts.

Patients with CKD had similar concentrations of TNF-α in the peripheral circulation and at the site of interstitial inflammation. By contrast, the concentration of TNF-α in the skin chamber was approximately five times higher than in the peripheral circulation in healthy controls. These differences in TNF-α concentrations and blood–interstitial gradients might contribute to the increased susceptibility towards infections generally associated with CKD. In support of this notion, an impaired gradient in TNF-α has been associated with an increased risk of septicaemia as well as an adverse outcome [35-38]. Although CD16+ monocytes produce TNF-α, the composition of inflammatory mediators in the skin chamber fluid is complex and results from activation of both extravasated as well as tissue dwelling cells.

Patients with CKD and healthy controls had similar concentrations of the anti-inflammatory IL-10 in the peripheral circulation. However, healthy controls, as opposed to patients with CKD, displayed a significant increase in IL-10 levels at the site of inflammation. This inability to regulate the inflammatory response by local production of IL-10 might add to the state of chronic systemic inflammation in patients with CKD as IL-10 is regarded as a crucial factor in suppressing the inflammatory response [39-41].

In 3–5 patients with CKD, monocytes have been shown to be more susceptible to apoptosis and display an higher proinflammatory activity [42, 43]. Our findings do not exclude these or any other kind of alterations in monocyte function.

In conclusion, patients with CKD display an increased proportion of the CD16+ monocytes both in the peripheral circulation and at a local site of induced interstitial inflammation. In addition, the present patients with CKD also displayed alterations in the immune-regulatory cytokines TNF-α and IL-10 in a manner that supposedly impairs the immune response, making these patients more susceptible towards infections and prone to chronic inflammation. The results shed light on processes influencing clinical dilemmas in CKD, such as the increased incidence of acute infections and the higher mortality from sepsis and pneumonia.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

We thank Anette Bygdén for skilful technical assistance with the skin chamber method. This study was supported by an unrestricted grant from the Karolinska Institutet, the Stockholm County Council, Stockholm, Sweden, and in part from Amgen Europe to SJ.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References