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(Journal of Leukocyte Biology. 2001;70:289-296.)
© 2001 by Society for Leukocyte Biology

Augmented TNF-{alpha} and IL-10 production by primed human monocytes following interaction with oxidatively modified autologous erythrocytes

Amy M. Liese*, Muhammad Q. Siddiqi*, John H. Siegel*, Thomas Denny{dagger} and Zoltán Spolarics*

Departments of
* Anatomy, Cell Biology & Injury Sciences, and
{dagger} Pediatrics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

Correspondence: Zoltán Spolarics, M.D., Ph.D., Associate Professor, UMDNJ-New Jersey Medical School, 185 South Orange Ave., MSB G-626, Newark, NJ 07103. E-mail: spolaric{at}umdnj.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of dysfunctional/damaged red blood cells (RBCs) has been associated with adverse clinical effects during the inflammatory response. The aim of this study was to elucidate whether oxidatively modified, autologous RBCs modulate monocyte cytokine responses in humans. Monocyte tumor necrosis factor {alpha} (TNF-{alpha}) and IL-10 production was measured in whole blood from healthy volunteers using ELISA and flow cytometry. Oxidatively modified RBCs (15 mM phenylhydrazine, 1 h, OX-RBC) or vehicle-treated RBCs (VT-RBC) opsonized by autologous serum were administered alone or in combination with one of three priming agents: E. coli lipopolysaccharide (LPS, 0.2 ng/ml), zymosan A (1 mg/ml), or phorbol 12-myristate 13-acetate (PMA, 50 ng/ml). OX-RBC or VT-RBC alone did not result in the release of TNF-{alpha} or IL-10. LPS, zymosan, and PMA caused marked and dose-dependent increases in TNF-{alpha} and IL-10 production. Addition of OX-RBC augmented the LPS-, zymosan-, and PMA-induced TNF-{alpha} release by approximately 100%. OX-RBC augmented LPS- and zymosan-induced IL-10 release by 400–600%. Flow cytometry analyses showed that monocytes were responsible for TNF-{alpha} and IL-10 production in whole blood. The presence of OX-RBC alone increased the complexity of CD14+ monocytes but caused no cytokine production. LPS alone induced cytokine production without altering cell complexity. After the combined (OX-RBC+LPS) treatment, monocytes of high complexity were responsible for TNF-{alpha} production. The presence of mannose or galactose (at 10–50 mM) did not alter the observed augmentation of cytokine production by OX-RBC, suggesting that lectin receptors are not involved in the response. These studies indicate that the interaction between damaged autologous erythrocytes and monocytes has a major impact on the cytokine responses in humans. An augmented cytokine production by the mononuclear phagocyte system may adversely affect the clinical course of injury and infections especially in genetic or acquired RBC diseases or after transfusions.

Key Words: red blood cells • cytokines • oxidative stress • macrophage • host response • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Red blood cell (RBC) interaction with macrophages has been well studied [1 2 3 ], and the role of RBCs in immune modulation has become better appreciated in recent years. RBCs have been demonstrated to bind opsonized immune complexes via complement receptor 1 (CR1) and to restrict immune complex uptake and subsequent degranulation by polymorphonuclear leukocytes [4 ]. Phagocytosis of immunoglobulin G (IgG)-coated RBCs by macrophages has been linked to decreased oxidative burst and inhibited bacterial killing [5 6 7 8 ]. Furthermore, RBC morphological changes and accompanying functional changes, including oxidative membrane alterations, decreased deformability, increased fragility, and accelerated aging, have been associated with septic complications and adverse clinical outcomes following trauma [9 10 11 ].

Atypical forms of RBCs are usually absent in blood because the mononuclear phagocyte system efficiently eliminates aged and/or damaged RBCs from the circulation. Under normal conditions, the elimination of RBCs is an "inert" process, i.e., it does not activate the mononuclear phagocyte system. However, in trauma patients with the Systemic Inflammatory Response Syndrome (SIRS) or sepsis, morphologically atypical RBCs are frequently present together with a markedly activated mononuclear phagocyte system [12 , 13 ]. These facts raise the question of whether the accumulation and subsequent elimination of damaged RBCs have an impact on the activation pattern of the mononuclear phagocyte system during an inflammatory response in humans.

The immune modulatory roles of RBC have been demonstrated in several animal investigations. It has been shown that IgG-coated sheep RBCs increased lipopolysaccharide (LPS)-induced tumor necrosis factor {alpha} (TNF-{alpha}) production by murine macrophages in vitro [14 ] and that IgG-coated rat erythrocytes increased serum TNF-{alpha} levels in rats in vivo [15 ]. Using a murine macrophage cell line and human RBCs, it was also demonstrated that phagocytosis of oxidatively damaged RBCs was increased over that of normal RBCs [16 ]. However, it remains unknown whether autologous RBC-macrophage interactions modulate the responses of the monocuclear phagocyte system in humans.

In the present study therefore we tested the hypothesis that the interaction between oxidatively challenged blood erythrocytes and monocytes alters the activation of monocytes in humans. Using autologous, experimental conditions, the effects of oxidatively modified RBCs were tested on nonactivated monocytes or on monocytes activated by nonspecific immune stimuli: Escherichia coli LPS, zymosan A, or following protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA). TNF-{alpha} and interleukin (IL)-10 production were determined as markers of monocyte activation. TNF-{alpha} and IL-10 were selected because they are essential mediators of the inflammatory response, and monocytes are important sources of these cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), zymosan A, PMA, phenylhydrazine hydrochloride, penicillin-streptomycin solution, brefeldin A, ethylenediaminetetraacetic acid (EDTA), and saponin were purchased from Sigma-Aldrich (St. Louis, MO). LPS (from E. coli O26:B6) was from Difco Laboratoires (Detroit, MI). Hank’s balanced salt solution (HBSS), without phenol red, and Dulbecco’s phosphate-buffered saline (PBS) were purchased from Life Technologies (Grand Island, NY). Methanol-free formaldehyde (10%) was purchased from Polysciences, Inc. (Warrington, PA). When applicable, cell-culture grade buffers, media, and reagents were used.

Blood samples and RBC treatments
This study was approved by the Institutional Review Board of New Jersey Medical School (Newark, NJ). Whole blood was collected from healthy, consenting adults. The investigated individuals represented eight males and four females with a mean age of 34.8 ± 2.3. Mean white blood cell counts were (thousand/µl blood): 6.7 ± 0.4 total white blood cells, 3.8 ± 0.2 neutrophils, 2.2 ± 0.2 lymphocytes, 0.47 ± 0.03 monocytes. Mean RBC count was 4.8 x 106 ± 0.1/µl blood. An aliquot of heparinized blood was diluted tenfold with pre-warmed DMEM containing 20 mM HEPES and penicillin-streptomycin solution, dispensed into 24-well tissue-culture plates, and kept at 37°C/5% CO2. The remaining heparinized blood was centrifuged at 150 g for collection of autologous RBCs. Sedimented RBCs were transferred to a fresh tube, washed once with warm HBSS, and treated as described below. Blood drawn without anti-coagulant was allowed to clot and was then subjected to centrifugation at 3500 rpm (2000 g) for 30 min at 4°C. Serum was collected and used for autologous opsonization of RBCs.

RBCs were incubated in the presence of 15 mM phenylhydrazine hydrochloride (OX-RBC) or HBSS vehicle (VT-RBC) for 1 h. Phenylhydrazine treatment has been well-established to cause oxidation of RBC membranes and the appearance of Heinz bodies [16 17 18 ]. After three washes, RBC suspensions were incubated with 50% autologous serum at 37°C for 30 min in HBSS. After the incubation, RBCs were washed in HBSS to remove serum, and the freshly prepared OX-RBC or VT-RBC was used for the treatments.

Treatments and cytokine assays
To minimize cell activation that is associated with isolation procedures, we used a whole blood cytokine assay as described previously [19 ]. Tenfold, diluted heparinized whole blood (2 ml final volume) was treated for 4–24 h at 37°C/5% CO2 with one of the following: vehicle (culture medium), LPS (0.04–100 ng/ml), zymosan (1 mg/ml), PMA (1–100 ng/ml), OX-RBC, or VT-RBC (0.2–4x105 RBC/µl final volume). This concentration range of OX-RBC represents an endogenous RBC:OX-RBC ratio of 20:1–1:1. OX-RBC and VT-RBC were also administered in combination with LPS or zymosan or PMA, respectively. Stimulating agents were added first, followed immediately by addition of VT-RBC or OX-RBC. Cell suspensions were incubated for 4, 6, 12, or 24 h. Following the incubations, supernatants were collected and stored at -80°C until initiating assay procedures. Samples were assayed for TNF-{alpha} and IL-10 using OptEIA kits (BD Pharmingen, San Diego, CA).

Flow cytometry
The number of TNF-{alpha}-producing monocytes in whole blood was determined by using fluorescein isothiocyanate (FITC)-labeled, anti-human CD14 (Becton Dickinson, San Jose, CA) and phycoerythrin (PE)-labeled, anti-human TNF-{alpha} (BD Pharmingen), as described previously [20 ] with modifications. Briefly, whole blood was incubated in the presence of vehicle (culture medium), LPS, OX-RBC, or the combination of LPS and OX-RBC in polypropylene tubes containing 10 µg/ml brefeldin A (a Golgi inhibitor) for 4 h at 37°C/5% CO2. Following the treatments, samples (2 ml) were incubated with 40 µl of 0.5 M EDTA for 30 min at 4°C followed by centrifugation at 300 g for 5 min. The supernatants were removed, and 150 µl sedimented cells were added to tubes pre-loaded with FITC-labeled, anti-human CD14. After gentle mixing, tubes were kept at room temperature in the dark for 15 min. RBCs were then lysed with 2.5 ml pre-warmed lysing solution (1.5 M NH4Cl/100 mM NaHCO3/10 mM EDTA) for 5 min and centrifuged at 300 g for 5 min. After two washes with azide wash buffer [0.5% fetal bovine serum (FBS)/0.1% sodium azide in PBS], pelleted leukocytes were permeabilized (0.1% saponin in % formaldehyde containing 10 mM HEPES) for 15 min at room temperature. Following two washes, cells were incubated with fluorescent-labeled, anti-human TNF-{alpha} for 30 min at room temperature. After washing, cells were fixed in 0.3 ml of 2% formaldehyde and kept at 4°C in the dark until acquisition. Analyses were performed using a FACScan flow cytometer and CellQuest software (Becton Dickinson, Mansfield, MA).

Visualization of monocyte-RBC interaction
Diluted whole blood subjected to various treatments was incubated at 37°C/5% CO2 for 4–24 h in polypropylene tubes. Following treatments, samples were centrifuged at 300 g for 5 min. The supernatant was removed, and sedimented cells were suspended in warm RBC-lysing buffer for 3 min. Cells were then washed twice with PBS, and 0.1 ml suspensions were centrifuged in duplicate for 3 min onto microscope slides using Cytospin 2 (Shandon Southern Instruments, Sewickley, PA) at 900 rpm. Cells were stained with Wright-Giemsa for light microscopy. Cell counts were performed on at least six random fields per slide.

Statistics
Statistical calculations were performed using JMP software (SAS Institute Inc., Cary, NC). Cytokine results were analyzed using analysis of variance (ANOVA) and Tukey-Kramer’s test for multiple comparisons. Statistically significant difference was concluded at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of oxidized RBC on cytokine release in whole blood
In pilot studies, we determined the concentration and time dependence of cytokine production in whole blood stimulated with LPS, zymosan, or PMA (using ELISAs). LPS increased TNF-{alpha} production dose-dependently at 0.04–100 ng/ml. Dose-dependent stimulation of IL-10 was observed at 0.2–100 ng/ml LPS. The maximal stimulatory doses of zymosan and PMA were 1 mg/ml and 50 ng/ml, respectively. TNF-{alpha} media content reached maximal levels at 6 h and remained unchanged for over 24 h. IL-10 levels were detectable at 12 h and reached plateau at 24 h. Based on these observations, subsequent experiments were performed after 24 h incubations.

Figure 1 shows the effect of OX-RBC, alone and in combination with one of the three priming agents, on cytokine production. TNF-{alpha} production was near the detection limit of the assay in samples treated with vehicle, VT-RBC, or OX-RBC. Treatment with a suboptimal dose of LPS (0.2 ng/ml) resulted in a 20-fold increase in TNF-{alpha} production (Fig. 1A) . Zymosan (1 mg/ml) stimulated TNF-{alpha} approximately 25-fold (Fig. 1B) , and PMA (50 ng/ml) stimulated TNF-{alpha} 30-fold (Fig. 1C) . When LPS, zymosan, or PMA was administered in the presence of OX-RBC, TNF-{alpha} release was increased by approximately 100% compared with the effect of the corresponding, individual priming agent alone (Fig. 1A 1B 1C) . The augmenting effect of OX-RBC on cytokine production was evident at 0.2–4 x 105 RBC/µl. In contrast to the effects of OX-RBC, VT-RBC did not alter TNF-{alpha} production in combination with any of the three stimulating agents.



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  		Figure 1. Effect of oxidatively modified RBC on TNF-{alpha} production in whole blood. TNF-{alpha} was measured following stimulation with vehicle (culture medium), VT-RBC or OX-RBC (2x105/µl), LPS (0.2 ng/ml), zymosan (1 mg/ml), PMA (50 ng/ml), or combinations of RBC with LPS, zymosan, or PMA. Cytokine levels were determined after 24 h incubation as described in Materials and Methods. Results are expressed as the mean ± SE normalized to monocyte number. Experiments were performed in duplicate on at least six different individuals. *, Statistically significant difference compared with LPS (A), zymosan (B), or PMA (C).

 
IL-10 was produced in low levels after treatment with vehicle, VT-RBC, or OX-RBC (Fig. 2 ). In contrast to the observed TNF-{alpha} release, IL-10 production was not increased significantly by treatment with 0.2 ng/ml LPS (Fig. 2A) . However, combined treatment with OX-RBC and LPS resulted in a marked fivefold increase in the production of IL-10 (Fig. 2A) . Basal IL-10 production was increased by approximately threefold after zymosan treatment and tenfold after the combined zymosan + OX-RBC treatment (Fig. 2B) . It is interesting that VT-RBC together with LPS or zymosan also enhanced the IL-10 response compared with LPS and zymosan effects alone. However, the effect of VT-RBC was significantly less than that of OX-RBC (Fig 2A and 2B) . PMA alone increased IL-10 release by fivefold (Fig. 2C) . The difference in IL-10 release between the combined PMA + OX-RBC and PMA alone treatment was not statistically significant (Fig. 2C) .



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  		Figure 2. Effect of oxidatively modified RBC on IL-10 production in whole blood. IL-10 was measured following stimulation with vehicle (culture medium), VT-RBC or OX-RBC (2x105/µl), LPS (0.2 ng/ml), zymosan (1 mg/ml), PMA (50 ng/ml), or combinations of RBC with LPS, zymosan, or PMA. Cytokine levels were determined after 24 h incubation as described in Materials and Methods. Results are expressed as the mean ± SE normalized to monocyte number. Experiments were performed in duplicate on at least six different individuals. *, Statistically significant difference compared with LPS (A), zymosan (B), or PMA (C). #, Statistically significant difference compared with LPS + VT-RBC (A) or zymosan + VT-RBC (B).

 
Flow cytometry analysis of whole blood cytokine production
Because ELISA measurements in whole blood do not provide information on which cells are responsible for the enhanced cytokine production, we determined the cellular source of TNF-{alpha} production using the intracellular cytokine-staining technique. Two-color flow cytometry was performed to simultaneously measure CD14-positivity and TNF-{alpha} production of peripheral blood leukocytes. Figure 3 shows a typical result after gating all white blood cells under nonstimulated conditions (Fig. 3A and 3C) or following incubation with LPS (Fig. 3B and 3D) . Under nonstimulated conditions, CD14-positive monocytes showed low levels of TNF-{alpha} staining (lower right quadrant of Fig. 3C ). Following LPS stimulation, CD14-positive monocytes showed a marked increase in TNF-{alpha} staining (upper right quadrant of Fig. 3D ). In contrast, no marked changes were observed after LPS stimulus in the granulocyte and lymphocyte fraction with low CD14-positive staining (upper left quadrants in Fig. 3C vs. D ). These data show that monocytes are the major sources of the LPS-induced production of TNF-{alpha} in whole blood. Similar observations were found after testing for the cellular source of IL-10 production in whole blood (unpublished results).



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  		Figure 3. CD14-postive monocytes are the predominant source of whole blood TNF-{alpha} production. Two-color flow cytometry analysis was used to simultaneously measure cell-associated TNF-{alpha} and CD14 in whole blood as described in Materials and Methods. Whole blood was incubated with vehicle (culture medium, left panels) or LPS (5 ng/ml, right panels) for 4 h. Upper panels show cell-associated CD14 fluorescence (x-axis) and side-scatter (y-axis). Lower panels show TNF-{alpha} and CD14 staining in dual plots. The position of quadrants was set according to the intensity of cell staining under nonstimulated conditions. One typical finding of several independent experiments is shown.

 
Figure 4 shows leukocyte side-scatter plots (measures of the cellular complexity) as a function of CD14-associated fluorescence. As shown, the CD14-positive cell population (monocytes) was divided into two gates. The lower gate includes monocytes with "low-complexity," and the upper gate shows cells with "high complexity." CD14-positive monocytes exhibited the low-complexity phenotype in vehicle-treated blood (Fig. 4A) or after LPS administration (Fig. 4C) . OX-RBC treatment markedly enhanced the "complexity" of monocytes independently, whether administered alone or in combination with LPS (Fig. 4B and 4D) .



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  		Figure 4. Effect of oxidatively modified RBC on monocyte complexity. Blood was stimulated with vehicle (culture medium; A), OX-RBC (2x105/µl) alone (B), LPS (0.2 ng/ml; C), or LPS + OX-RBC (D) for 4 h. Panels depict typical findings from four independent determinations. Boxed areas encase CD14-positive cells. Lower boxes include monocytes of low complexity. Upper boxes include monocytes of high complexity.

 
Because side scatter is also influenced by structural complexity, granularity, and potentially by cell-cell interactions [21 , 22 ], we tested RBC-monocyte interactions following these treatments. In the presence of VT-RBC, 10.3 ± 4.9% of monocytes were associated with RBCs, whereas in the presence of OX-RBC, 89.2 ± 1.9% of monocytes were associated with RBCs (Fig. 5A and C ). Addition of LPS had no measurable effects on monocyte-RBC interactions (Fig. 5B and 5D) . These data are consistent with the flow cytometry findings, indicating that monocyte-RBC interactions may contribute to the observed increase in cell complexity.



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  		Figure 5. Association of oxidatively modified RBC with monocytes. Diluted whole blood was incubated with autologous VT-RBC alone (A), LPS (0.2 ng/ml) + VT-RBC (B), OX-RBC alone (C), or LPS (0.2 ng/ml) + OX-RBC (D) for 24 h. Cells were visualized by light microscopy as described in Materials and Methods. A typical finding of several experiments is shown.

 
Table 1 displays the effect of LPS (0.2 ng/ml) and OX-RBC on the distribution of monocytes according to complexity and TNF-{alpha}-positivity. OX-RBC treatment resulted in a threefold increase in the number of high-complexity monocytes without an increase in the number of TNF-{alpha}-positive cells. After LPS treatment, two-thirds of blood monocytes stained positive for TNF-{alpha}, and nearly all of the cells showed low complexity. After the combined treatment (LPS+OX-RBC), two-thirds of the monocytes showed high complexity, and the majority of these cells were also TNF-{alpha}-producing. Approximately 60% of all monocytes stained positive for TNF-{alpha} after LPS or the combined LPS + OX-RBC treatments (Table 1) . Administration of VT-RBC had no effect on monocyte complexity or TNF-{alpha} production (unpublished results).


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  		Table 1. Flow Cytometry Analysis of the Distribution of TNF-{alpha} Production by CD14-positive Cell Subpopulations

 
It was shown previously that recognition and phagocytosis of damaged RBCs are mediated by macrophage lectin-like receptors [16 , 23 , 24 ]. In pilot studies, we tested whether masking lectin-like receptors by administration of mannose and galactose would alter the OX-RBC-induced cytokine production. We found that the presence of mannose or galactose (up to 50 mM each) or the combination of the two had no effect on the LPS + OX-RBC-, zymosan + OX-RBC-, or PMA ± OX-RBC-induced increases in TNF-{alpha} or IL-10 production (unpublished results).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These studies demonstrate, for the first time, that under autologous experimental conditions, the presence of oxidatively stressed erythrocytes in blood exacerbates cytokine production markedly and thus, the activation status of human monocytes. The presence of oxidatively stressed erythrocytes did not activate resting monocytes, however it markedly enhanced the cytokine response in monocytes primed by distinct, inflammatory response modifiers.

During an inflammatory response, damaged RBCs as well as activated mononuclear phagocytes are present in the tissues and in the circulation. Free radicals from phagocytes targeting RBCs or generated within the cell may contribute to oxidative-membrane alterations of RBCs [25 , 26 ]. Increased RBC membrane-lipid viscosity, increased membrane-lipid peroxidation, and decreased RBC deformability, which were associated with the development of multiple-system organ failure [10 , 27 , 28 ], were observed in septic patients In experimental rodent models, trauma resulted in RBC modifications that altered hemodynamic function [29 ], and the mortality rate caused by endotoxin and bacterial infection was shown to be increased by ingestion of RBC by macrophages [8 ]. Conversely, excessive macrophage activation and the production and release of cytokines following trauma or infection were shown to initiate tissue injury and organ dysfunction [13 , 30 31 32 ]. Our current study suggests that an augmented cytokine production by monocytes and presumably also by resident tissue macrophages may be an important cellular mechanism that contributes to the described aggravation of the inflammatory response in the presence of damaged, circulating RBCs.

Trauma patients, as well as individuals with chronic or acute RBC disorders, are commonly the recipients of blood transfusions. Previous studies by others have demonstrated that production of inflammatory cytokines by phagocytes may be responsible for the symptoms related to hemolytic transfusion reactions. Davenport et al. [33 ] showed that nonautologous RBCs that had been sensitized with anti-D IgG evoked an elevated cytokine release (IL-1ß, IL-6, IL-8, and TNF-{alpha}). In general, these data are in agreement with our finding that RBCs, when modified or sensitized, may modulate human monocyte function. However, our observations indicate that RBCs do not require foreign antibody opsonization to trigger their effects. Our results may also provide an explanation as to why large-volume RBC transfusions (especially older preparations) may aggravate the inflammatory response in trauma patients.

The signaling mechanism involved in the observed OX-RBC-induced TNF-{alpha} production by activated monocytes remains unclear. Despite the fact that the three priming agents used in our study initiate monocyte activity by targeting distinct membrane components, OX-RBC stimulated TNF-{alpha} production quite similarly when used together with any of these agents. LPS initiates TNF-{alpha} production via a CD14 receptor-mediated signaling cascade. This has been shown to involve LPS-binding protein, toll-like receptor 4, activation of protein kinases, and the activation of transcription factors including nuclear factor-{kappa}B (NF-{kappa}B) [34 35 36 37 ]. Zymosan is recognized at the monocyte mmbrane by complement receptor 3 (CR3, ß2 integrin CD11b/CD18) [38 ]. Although it has been established that engagement of monocyte CR3 leads to NF-{kappa}B activation and production of inflammatory mediators [39 40 41 ], specific signaling mechanisms regulating cytokine production by zymosan are still unknown. PMA, a potent and direct activator of PKC, is shown to stimulate the production of inflammatory cytokines via phosphorylation of mitogen-activated protein kinases (MAPKs) and activation of the transcription factors AP-1 and NF-{kappa}B [42 , 43 ]. Based on the fact that the exacerbation of TNF-{alpha} production by OX-RBC was similar after each of the combined treatments, we suggest that the responsible signaling mechanism is downstream of events occurring at the monocyte plasma membrane. It remains to be tested whether the interaction between OX-RBCs and monocytes results in the activation of PKC, MAPKs, or NF-{kappa}B, which are downstream regulatory events in the LPS- and zymosan-induced responses [44 45 46 ].

The OX-RBC-induced augmentation of IL-10 production was more pronounced than TNF-{alpha}. Furthermore, the presence of VT-RBC in combination with LPS or zymosan also increased IL-10 production, although at a lesser degree than OX-RBC. These observations indicate that the in vitro manipulation and opsonization of RBC may have initiated RBC changes that promoted IL-10 production by primed monocytes. These observations also suggest that the mechanism of the augmenting effects responsible for TNF-{alpha} and IL-10 production may be different.

The facts that galactose and mannose did not influence the OX-RBC-induced, enhanced TNF-{alpha} production indicate that RBC-monocyte interactions via lectin receptors [16 , 23 ] are not required for the observed augmentation of cytokine response. A variety of mechanisms have been proposed to participate in RBC-macrophage interactions, including naturally occurring IgG or complement receptors [1 , 2 ]. The exact mechanism that is responsible for the observed augmentation of cytokine production by damaged RBCs in humans remains to be elucidated using serum- and plasma-free experimental systems.

Previous investigations showed that murine–macrophage interaction with IgG-coated RBCs results in an increase of de novo TNF-{alpha} production [14 ]. In our flow cytometry assays, the total number of TNF-{alpha}-producing cells was similar after treatment with LPS alone and after the combined LPS + OX-RBC treatments. This suggests that the augmented release of TNF-{alpha} in the whole blood cell cultures was the result of an elevated TNF-{alpha} production by monocytes and not an increase in the number of TNF-{alpha}-producing cells.

The fact that activated monocytes stimulated with oxidized RBCs produced augmented levels of TNF-{alpha} and IL-10 suggests their prolonged activation. TNF-{alpha}, widely accepted as being one of the body’s primary and early mediators of inflammation [47 ], was measured in this study as a marker of monocyte pro-inflammatory activity. IL-10 is recognized predominantly as a counter-inflammatory cytokine [32 , 48 ]. In our system, and consistent with the findings of others [49 ], IL-10 was detected later than TNF-{alpha}. Although it cannot be discounted that the observed IL-10 production was a result of autocrine stimulation by TNF-{alpha}, nevertheless, the marked increase in IL-10 production suggests a general and sustained increase in the activation status of monocytes in the presence of damaged erythrocytes. The elevated IL-10 production observed in our study after RBC-monocyte interaction may contribute to the down-regulation of oxidative burst and bactericidal activity by macrophages, which was demonstrated following the engulfment of senescent/damaged RBCs [6 7 8 , 50 ].

It has been shown that high concentrations of free hemoglobin or met-hemoglobin can stimulate cytokine production in macrophages [51 , 52 ]. Hemoglobin or met-hemoglobin may be released from stressed RBCs. However, the facts that the presence of OX-RBC alone had no effect on cytokine production and the augmenting effect of OX-RBC was manifested after using different priming agents make it unlikely that free hemoglobin or LPS-hemoglobin interactions [53 ] contributed to the observed responses.

Taken together, our data show that interaction between monocytes and oxidatively modified RBCs may have an important impact on the host response to trauma and infection by augmenting the cytokine responses of the mononuclear phagocyte system. The observations suggest that interactions of damaged RBCs with activated members of the mononuclear phagocyte system may contribute to the aggravation of the clinical status in sepsis or multiple organ-dysfunction syndromes. These data also imply that the clinical course of infections and trauma may be worsened in the presence of erythrocytes with an increased sensitivity to oxidative or other stresses in patients with acquired or genetic RBC diseases such as glucose-6-phosphate dehydrogenase deficiency, sickle cell anemia, and thalassemias or following blood transfusions.


   ACKNOWLEDGEMENTS
 
This study was supported by NIH-NIGMS grant GM-55005. We thank Zenaida C. Garcia and Dana S. Stein for their technical contributions in the flow cytometry measurements.

Received February 17, 2001; revised May 4, 2001; accepted May 4, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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