INTRODUCTION

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Peripheral blood monocytes are members of the mononuclear phagocytic system, which plays a central role in immunoregulation and host defense against immunopathogenic organisms (7). Monocytes are activated through molecular signals provided by structures of the infective organisms (8, 27, 28, 34, 35) or inflammatory mediators and chemotactic factors released by other cells during the infective challenge (22, 44, 47). However, blood monocytes represent a heterogeneous cell population and can be distinguished by variations in morphology (38, 58), membrane antigen expression (39), and release of inflammatory mediators (12, 25, 41).

While the lipopolysaccharide (LPS) receptor antigen CD14 is expressed by nearly all circulating peripheral blood monocytes, monocytes differ markedly in cell surface CD14 density as well as in the expression of immunoglobulin Fc receptors (53, 67). The majority of monocytes strongly positive for CD14 (CD14⁺⁺) express Fc receptor I (CD64) and Fc receptor II (CD32) and are negative for Fc receptor III (CD16) (18). Only a small population was identified by the absence of Fc receptors (63). Nevertheless, a subset of monocytes characterized by low-level expression of CD14 and expression of the CD16 antigen has also been described (40). In healthy subjects these CD14⁺ CD16⁺ cells account for about 10% of all monocytes and are thought to be more mature cells than the regular CD14⁺⁺ monocytes, as they exhibit features of tissue macrophages (66). In various infectious or inflammatory diseases such as AIDS and asthma the CD14⁺ CD16⁺ monocyte subpopulation is markedly expanded (36, 43, 50). A more than 10-fold increase of these cells during septicemia was demonstrated, and CD14⁺ CD16⁺ cells become the predominant type of monocytes in some septic patients (14).

Patients with end-stage renal failure undergoing chronic hemodialysis (HD) show an impaired immune response (10) with a high prevalence of infectious complications (17). Most of these infections are of bacterial origin, representing a major cause of morbidity and mortality in chronic HD patients (24). Furthermore, acute or chronic inflammatory processes, among them pneumonia and vascular access site infections, are common hazards in uremic patients undergoing chronic regular HD. Despite some data on the functional abnormalities of polymorphonuclear leukocytes in uremia (19), little information exists on the level of monocytes and their subsets in maintenance dialysis patients.

In an effort to further understand the importance of the distinct monocyte population expressing Fc receptor type III, we determined the levels of these cells in patients with end-stage renal failure undergoing chronic HD. This allowed the level of CD14⁺ CD16⁺ cells to be compared to that of CD14⁺⁺ cells and the total monocyte count in whole blood. To investigate the proinflammatory role of CD14⁺ CD16⁺ monocytes, stable patients as well as patients with acute or chronic signs of infections or inflammatory processes were studied. Furthermore, we analyzed cell surface HLA expression of CD14⁺ CD16⁺ monocytes by immunophenotyping and compared their phagocytic competence with that of regular CD14⁺⁺ blood monocytes.

	MATERIALS AND METHODS

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Patients. The patient population was divided into the following groups: informed outpatients on chronic maintenance HD with stable disease and no clinical or laboratory signs of an infectious episode (n = 18) and age- and sex-matched HD patients suffering from chronic infectious diseases (n = 16). Among the patients monitored over a period of 6 months, those patients with recurrent infections were analyzed in more detail during the active infectious episodes. Such episodes were defined by one or more of the following conditions: increase in C-reactive protein (CRP), borderline leukocytosis and fever, leukocytosis, inflamed area of vascular access with fever and increase of acute-phase reactants, positive blood culture data, positive histological evaluation (e.g., tuberculosis), recurrent fever episodes due, e.g., to chronic bowel infection (subileus or pancreatitis; increase in serum amylase and lipase activity), repeated fever due to underlying systemic disease (AA-type amyloidosis) or chronic bronchopneumonia (radiological signs of infiltrations), cirrhosis of the liver and intermittent inflammatory signs, recurrent ophthalmitis, peripheral arterial occlusive disease with ulcers, and positive bacterial cultures of cutaneous smears. In addition chronic viral infections (human immunodeficiency virus [HIV] or hepatitis A, B, or C) were monitored. Table 1 summarizes the diagnoses, as well as clinical and laboratory data, of the HD patients with chronic or recurrent infections studied.

Blood sampling. For counting blood monocytes and determining their cell surface antigen expression, whole blood was collected in tubes containing EDTA-Monovetten (Sarsted, Nümbrecht, Germany). Blood specimens were prepared for flow cytometry within 30 min after venipuncture. Absolute numbers of monocytes were calculated by using leukocyte counts derived from an automated blood cell counter (Coulter Electronics, Hialeah, Fla.).

MAbs and monocyte labelling. The following murine monoclonal antibody (MAb) clones used for phenotyping peripheral blood monocytes were purchased from Becton Dickinson (Heidelberg, Germany): Fluorescein isothiocyanate (FITC)-conjugated anti-CD14, phycoerythrin (PE)-conjugated anti-CD14, anti-CD16 (Fc receptor III), and PerCP-conjugated anti-HLA-DR; those purchased from Pharmingen (Hamburg, Germany) were the following: FITC-conjugated anti-HLA-DP, anti-HLA-DQ, and anti-HLA-ABC; that purchased from Immunotech (Hamburg, Germany) was PE/Cy5-conjugated anti-CD16. Fluorochrome-labelled anti-immunoglobulin G1 (IgG1) and IgG2 isotype control antibodies were purchased from Becton Dickinson.

Flow cytometry analysis. Flow cytometric analysis was performed with a FACScan flow cytometer (Becton Dickinson). Monocytes were first gated according to their forward- and side-scatter profiles, and then FITC, PE, PerCP, or PE/Cy5 channel fluorescence was measured within the monocyte gate. For the determination of specific antigen expression, the differences in fluorescence intensity between a specific MAb stain and the control stain were calculated. Results were expressed as mean fluorescence intensity (MFI).

Measurement of phagocytic activity. The quantitative determination of leukocyte phagocytosis in whole blood was performed with the Phagotest kit (Orpegen Pharma, Heidelberg, Germany). The percentages of monocytes and granulocytes showing phagocytosis as well as the amount of phagocytic activity per cell were analyzed. The performance of this test was modified in order to analyze different monocyte subpopulations according to their levels of membrane antigen expression. Briefly, for each specimen two test tubes were filled with 100 µl of heparinized blood and precooled on ice for 10 min before 20 µl of FITC-labelled Escherichia coli bacteria (2 × 10⁸ cells) was added. The control samples remained on ice, whereas assay samples were incubated for exactly 10 min at 37 ± 0.5°C in a water bath. At the end of incubation all samples were placed on ice, and 100 µl of quenching solution was added to suppress the fluorescence of adhering and noningested bacteria. After being washed with phosphate-buffered saline, monocytes were stained with a PE-conjugated anti-CD14 MAb and a PE/Cy5-conjugated anti-CD16 MAb for 15 min at 4°C. Then the whole blood was lysed, fixed with a lysis solution containing 1% paraformaldehyde as a fixative, and washed again.

Statistics. All statistical analyses were performed with the software package BIAS 5.0 from H. Ackermann, Department of Biomathematics, University of Frankfurt, Frankfurt am Main, Germany. Student's t test was used to estimate the statistical significance of the results. Data are expressed as means ± standard deviations (SD) and were considered statistically significant if P values were <0.05. Spearman's rank correlation test was applied to evaluate possible correlations between different study parameters within a group.

	RESULTS

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Distribution of CD14⁺ CD16⁺ monocytes in healthy subjects and patients under HD. Leukocytes stained with anti-CD14 and anti-CD16 MAbs revealed two subsets of peripheral blood monocytes in human blood (Fig. 1). Whereas nearly all monocytes were positive for the CD14 antigen, differences were found with regard to coexpression of the CD16 antigen. One population expressed high levels of CD14 (CD14⁺⁺) and accounted for the majority (92% ± 4%) of CD14⁺ monocytes, with an absolute number of 336 ± 134 cells/µl in healthy donors (Table 2). The second population expressed lower levels of CD14 together with the CD16 antigen. These CD14⁺ CD16⁺ monocytes constituted a minor population, i.e., about 8% ± 4% of CD14⁺ monocytes in healthy subjects (Fig. 1A), with an absolute number of 29 ± 14 cells/µl.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   CD14 and CD16 staining of peripheral blood monocytes. Whole-blood specimens from a healthy donor (A) and an HD patient (B) were stained with FITC-conjugated anti-CD14 and PE-conjugated anti-CD16 antibodies. Monocytes were gated by their light scatter profiles, and within the monocyte population the fluorescence gates of CD14-FITC and CD16-PE were set as indicated. Lower right gate, CD14++ monocytes; upper right gate, CD14+ CD16+ monocytes. The CD14+ CD16+ monocyte subset percentages were 7% (A) and 20% (B).

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Comparison of the percentages of CD14+ CD16+ monocytes and total monocyte counts. Whole-blood specimens were obtained from healthy controls () and patients under chronic HD (). Total blood monocyte counts were determined by an automated blood cell count, and the distribution of CD14+ CD16+ monocytes was calculated by flow cytometry after staining with anti-CD14 FITC and anti-CD16 PE. No correlation was found between the two parameters, either in HD patients or in healthy controls.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Intraindividual variation of CD14+ CD16+ monocytes in five patients undergoing chronic intermittent HD. The percentages of CD14+ CD16+-coexpressing cells were determined before an HD session by fluorescence-activated cell sorter analysis once weekly for 2 months.

Increase of the CD14⁺ CD16⁺ monocyte population during acute infections. During phases of acute infectious episodes the CD14⁺ CD16⁺ monocyte subpopulation in HD patients was found to be markedly expanded (Fig. 4A), whereas changes in membrane CD14 density on peripheral blood monocytes were not uniform (Fig. 4B). However, changes in CD14 surface expression were parallel in CD14⁺⁺ and CD14⁺ CD16⁺ monocyte populations. In contrast, in six febrile HD patients (38.2 to 39.5°C) the increment of CD14⁺ CD16⁺ monocytes could be correlated with clinical and serological findings such as leukocytosis, increase of CRP, and positive blood culture. In four HD patients with acute illness, bacterial infections were found to be causative. In three cases staphylococci were involved (shunt infection, infected jugular vein catheter, infected right atrial catheter). Another patient suffered from acute tracheobronchitis (Klebsiella pneumoniae), fluid in the lung, and uveitis. Two other patients suffered either from acute cholangitis, enteritis, and subileus or acute bronchopneumonia (coughing, mucopurulent expectoration, lung infiltrations, respiratory alkalosis). In both cases blood cultures were positive for gram-negative bacteria. The first patient had a serum alkaline phosphatase level of 420 U/liter and a -glutamyl transpeptidase level of 267 U/liter; ultrasound investigation of the bowel disclosed a sludge phenomenon of the gall bladder and slightly enlarged intrahepatic bile ducts. The second patient had a homogeneous nonsegmental consolidation in the right and left lower lobes and small parapneumonic effusions of both sinus pleurae.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Changes of monocyte CD14+ CD16+ subset (A) and membrane CD14 (B) expression during acute infections. Values are shown for six HD patients during the acute infectious phase (during) and compared with previous data measured during routine examinations 1 to 14 days before the onset of clinical symptoms (before). Each patient is represented by the same symbol in panels A and B. For details of the clinical course see the text. In panel B, CD14++ monocytes are represented by solid symbols and CD14+ CD16+ monocytes are represented by open symbols.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Expanded CD14+ CD16+ monocyte population in a patient with catheter sepsis. Blood monocytes were stained and analyzed by flow cytometry. For details of the clinical course see the text. The plus sign indicates the time of blood culture positive for S. aureus.

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Monocyte CD14+ CD16+ subpopulation and HLA-DR expression in a patient during enterococcus sepsis. CD14+ CD16+ monocyte levels and HLA-DR surface densities were determined by flow cytometry. Leukocytosis, 18,000 cells/µl. Details of the clinical course are presented in the text. The plus signs indicate the times of blood cultures positive for enterococci.

Phagocytic activities of CD14⁺⁺ and CD14⁺ CD16⁺ blood monocytes. Phagocytosis of microorganisms or particles is one of the most important functions of phagocytic cells during an infective challenge. We therefore compared the phagocytic activities of granulocytes, CD14⁺⁺ monocytes, and CD14⁺ CD16⁺ monocytes from 14 healthy donors by using opsonized FITC-labelled bacteria in a whole-blood assay.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Phagocytic activities of blood leukocyte populations. Phagocytosis of FITC-labelled E. coli bacteria was measured in a whole-blood assay by flow cytometry. Blood aliquots were either incubated at 0°C as controls or at 37°C to initiate phagocytosis. Monocytes and granulocytes were gated according their light scatter profiles, and monocyte subpopulations were determined by anti-CD14-PE and anti-CD16-PE/Cy5 immunofluorescence staining. Phagocytosis was determined by measuring E. coli FITC fluorescence, which correlates with the number of ingested bacteria. The figure shows phagocytosis rates of CD14++ monocytes, CD14+ CD16+ monocytes, and granulocytes from a representative example.

View larger version (22K): [in this window] [in a new window]   FIG. 8.   Comparison of leukocyte phagocytic activity in individuals. Phagocytosis of FITC-labelled E. coli bacteria in blood leukocytes from healthy donors was determined as indicated in the legend for Fig. 7. Data from 12 of 14 whole-blood specimens are shown. Leukocyte populations (CD14++ monocytes, CD14+ CD16+ monocytes, and granulocytes) from each donor are represented by the same symbol. Results are expressed as values of FITC MFI, which correlates with the number of bacteria ingested by each leukocyte population.

MHC antigen expression of CD14⁺⁺ and CD14⁺ CD16⁺ cells. The cell surface density of major histocompatibility complex (MHC) antigen expression by monocyte subpopulations was analyzed by three-color immunofluorescence. The results of the quantitative determination are displayed in Table 3. CD14⁺ CD16⁺ monocytes expressed significantly higher levels of MHC class II antigens. HLA-DR and HLA-DP expression was fourfold and about threefold higher, respectively, in these cells than in CD14⁺⁺ monocytes. Also, the membrane expression of the HLA-DQ antigen was found to be increased in CD14⁺ CD16⁺ monocytes, but the difference in antigen density was not so pronounced. No difference in MHC class I expression was observed; CD14⁺ CD16⁺ and CD14⁺⁺ monocytes expressed similar amounts of HLA-ABC antigens.

	DISCUSSION

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Several important functions during an infectious challenge, e.g., phagocytosis, intracellular killing of ingested microorganisms, and immunoregulatory activities, are performed by cells of the mononuclear phagocyte system. Therefore, different subsets of peripheral blood monocytes may play different and pivotal roles during the immune response. Recently, two subsets of peripheral blood monocytes with functional differences in bacterial uptake and antigen processing of Listeria monocytogenes were identified (64).

In the past many monocyte antigens that serve as receptors for recognition and processing of bacterial antigens have been described. The CD14 membrane antigen functions as a receptor for LPS from gram-negative bacteria (52, 56, 61) and triggers LPS-induced monocyte activation (29). Moreover, CD14 seems to be involved in the recognition of structures from gram-positive bacteria such as lipoteichoic acid and peptidoglycan (6, 59). In addition, receptors for the Fc region of IgG are important in the phagocytosis of IgG-opsonized microorganisms (53, 54). Fc receptor type III (CD16) exists in two polymorphic forms: the transmembrane form (FcRIIIa) expressed in monocytes/macrophages and the glycosyl phosphatidylinositol-anchored form (FcRIIIb) expressed in granulocytes (53, 54). The FcRIIIa form on monocytes is reported to mediate important immunophysiological functions such as superoxide generation (51) and antibody-dependent cell mediated cytotoxicity (13), and signal transduction by Fc receptor type III is mediated through the  chain of the Fc receptor type III complex (33, 60). It is known that immune complexes cross-link Fc receptors on the surfaces of monocytes and induce tumor necrosis factor secretion (9). Fc receptor type III has little affinity for monomeric IgG but binds polymeric IgG or immune complexes efficiently (5). Blockade of this receptor by an anti-CD16 MAb inhibits the clearance of opsonized cells in vivo, indicating the influence of this membrane receptor in the removal of immune complexes during infections (4).

While in the past peripheral blood monocytes were thought to be negative for CD16 and to upregulate this antigen during culture in vitro (5, 26), a minor subpopulation of circulating monocytes which express Fc receptor type III together with smaller amounts of CD14 has been described (65). The CD14⁺ CD16⁺ monocyte subpopulation is significantly expanded in patients with severe infections (14) and can account for more than 50% of all peripheral blood monocytes during septicemia. In the present study the percentage of CD14⁺ CD16⁺ monocytes was found to be significantly higher in HD patients than in healthy controls, with a further increase in patients suffering from chronic infectious or inflammatory diseases. These findings further support the importance of the CD14⁺ CD16⁺ monocyte subpopulation during an infectious challenge.

It is well known that HD patients have an increased susceptibility to infectious diseases, and infections account for 30 to 40% of deaths in patients on long-term HD (32). These infections are generally due to common and not opportunistic pathogens (23), but mycobacterial infections and tuberculosis are also more frequent in HD patients than in healthy subjects (3, 21). The diagnosis of tuberculosis is often difficult to establish because the symptoms may mimic those of uremia (1), and thus infections with Mycobacterium tuberculosis with low-grade disease activity may be clinically symptomless. Recently, an elevated percentage (tripled compared to controls) of CD14⁺ CD16⁺ monocytes from pulmonary tuberculosis patients has been described (55), underlining the possible diagnostic value of the expanded CD14⁺ CD16⁺ cell population in HD patients with potential mycobacterial infections.

Viral diseases such as hepatitis B and C and cytomegalovirus infection are also common in dialysis patients; these contribute to the impaired immune response and increase the risk of further secondary infections. In addition, patients who receive dialysis therapy are under the continuous threat of infections from vascular or peritoneal access, which is clearly demonstrated by the case of the chronic ambulatory peritoneal dialysis patient described above. This issue has been discussed in detail by other investigators (48).

Recent studies reported expanded populations of CD14⁺ CD16⁺ monocytes in HIV-infected patients also prone to recurrent infectious crises (31, 36, 50). Together with the present observations for HD patients and those from other workers for sepsis patients (14), these studies show that the CD14⁺ CD16⁺ monocyte subpopulation plays an important role during an infectious challenge. This is consistent with the higher surface density of MHC class II molecules which are necessary for antigen presentation to T cells. There is growing evidence that the expression of each type of MHC class II molecule on human monocytes is regulated independently of the others (46, 62). Thus, increased expression of three different HLA class II clusters (DR, DQ, and DP antigens) by CD14⁺ CD16⁺ cells underlines their role as potent antigen-presenting cells. Furthermore, the cell surface densities of adhesion molecules such as LFA-1 and ICAM-1 known to be involved in leukocyte cell-cell interactions are higher in CD14⁺ CD16⁺ than in CD14⁺⁺ monocytes (66).

Using PCR analysis Frankenberger et al. (15) showed that CD14⁺ CD16⁺ cells expressed levels of mRNA for the proinflammatory cytokines interleukin 1 (IL-1), IL-6, and tumor necrosis factor similar to those expressed by CD14⁺⁺ monocytes. In contrast, the level of mRNA for the potent anti-inflammatory cytokine IL-10 (11, 49) was substantially lower or undetectable in CD14⁺ CD16⁺ cells (15). This altered cytokine expression together with the higher cell surface densities of MHC class II antigens indicates that CD14⁺ CD16⁺ monocytes represent a potent antigen-presenting and proinflammatory subpopulation, possibly triggering immune responses during an infectious challenge.

There are few parameters that can be useful for monitoring the host response and for predicting the progression of chronic and acute infections. The relevance of determinations of serum concentrations of inflammatory cytokines and other substances released during the activated immune response is still under investigation (2, 16). Monitoring the cellular immune system in patients with increased incidence of infections seems to be another important approach to develop strategies for clinical intervention (20, 42, 57). Here, peripheral blood monocytes and their HLA-DR expression have been studied (30). However, determination of the expression of the highly phagocytosing and antigen-presenting CD14⁺ CD16⁺ monocyte subset, which is expanded in various inflammatory and infectious diseases, may also be helpful.