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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Systemic lupus erythematosus (SLE) is characterized by intravascular activation of the complement system and deposition of complement fragments (C3 and C4) on plasma membranes of circulating cells, including red blood cells (RBCs). The aim of this study was to address whether this process affects the biophysical properties of RBCs.

Methods

Serum and RBCs were isolated from patients with SLE and healthy controls. RBCs from healthy universal donors (type O, Rh negative) were incubated with SLE or control serum. We used flow cytometry to assess complement fragment deposition on RBCs. RBC membrane deformability was measured using 2-dimensional microchannel arrays. Protein phosphorylation levels were quantified by Western blotting.

Results

Incubation of healthy universal donor RBCs with sera from patients with SLE, but not with control sera, led to deposition of C4d fragments on the RBCs. Complement-decorated RBCs exhibited significant decreases in both membrane deformability and flickering. Sera from SLE patients triggered a transitory Ca++ influx in RBCs that was associated with decreased phosphorylation of β-spectrin and with increased phosphorylation of band 3, two key proteins of RBC cytoskeleton. Finally, incubation with SLE sera led to the production of nitric oxide by RBCs, whereas this did not occur with control sera.

Conclusion

Our data suggest that complement activation in patients with SLE leads to calcium-dependent cytosketeletal changes in RBCs that render them less deformable, probably impairing their flow through capillaries. This phenomenon may negatively affect the delivery of oxygen to the tissues.

During systemic lupus erythematosus (SLE) flares, circulating immune complexes lead to intravascular complement consumption and deposition of nascent C3 and C4 fragments on red blood cell (RBC) membranes (1–3). Although this phenomenon is well established, very little is known about the effect of complement fragment deposition on RBC physiology in SLE.

In a recent study, our group showed that deposition of C3 and C4 complement fragments on RBCs in vitro promoted a significant decrease in RBC membrane deformability (4), which influences the ability of RBCs to change shape in response to external forces (5, 6). When RBCs enter the microcirculation where the gas exchange takes place, the cell diameter changes rapidly, from 7.5–8 μm to 4.5–5 μm. The dynamic, energy-dependent link between cell membrane and skeleton is essential for the ability of RBCs to pass rapidly through capillaries without cell fragmentation, and critical for maintenance of adequate tissue perfusion (7, 8).

Based on our previous in vitro findings, we hypothesized that deposition of complement fragments on circulating RBCs in SLE leads to impaired RBC deformability. Here we present evidence for the first time that incubation of RBCs from healthy donors with sera from SLE patients results in complement fragment deposition, significant changes in the phosphorylation status of skeletal proteins, and a decrease in RBC membrane deformability. Moreover, RBCs with membrane-bound complement produce increased amounts of nitric oxide (NO), an important mediator of pathophysiologic processes characterizing the immune system dysregulation in SLE.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Subjects.

Thirty-nine patients who fulfilled the American College of Rheumatology revised criteria for the classification of SLE (9) and 12 healthy donors donated 20 ml of blood for our studies (53 unique blood collections from SLE patients and 17 from controls). The serum was collected in serum-separator tubes, and RBCs were collected in heparin–lithium tubes. The patient group consisted of 37 women (95%) and 2 men, with a mean age of 37.5 years (range 23–59 years). The controls were all female and had a mean age of 35 years (range 20–52 years). Fifty-six percent of the SLE patients and 58% of the controls were white; 23% of the SLE patients and 25% of the controls were African American, and 20% of the SLE patients and 17% of the controls were of other ethnic origin.

Disease activity was calculated using the SLE Disease Activity Index (SLEDAI) (10). Sixty-eight percent of the patients were taking oral prednisone, at a mean dosage of 23 mg/day (range 2–60). Prior to blood drawing, 1 patient received 1 dose of intravenous methylprednisolone (1,000 mg). Other immunosuppressive medications patients were receiving at the time of the study included hydroxychloroquine (66%), azathioprine (19%), and mycophenolate mofetil (30%). One patient was receiving intravenous monthly cyclophosphamide, 1 patient was taking cyclosporine, and 3 patients were taking weekly methotrexate at the time of the study. Prednisone was stopped for at least 12 hours prior to blood drawing. The Institutional Review Board of Beth Israel Deaconess Medical Center approved the study protocol, and informed consent was obtained from all of the study subjects.

Antibodies and reagents.

Primary antibodies were nonimmune IgG1 (BD Biosciences), anti-C4c monoclonal antibody (mAb) (A211; Quidel), anti-CR1 mAb 1F11 (a gift from Henry Marsh, PhD, Celldex Immunotherapeutics, Needham, MA), and antiactin and antiphosphoserine mAb (both from Abcam). Secondary antibodies were Alexa Fluor 488–conjugated goat anti-mouse IgG, Alexa Fluor 594–conjugated goat anti-rabbit IgG, and Alexa Fluor 594–conjugated goat anti-mouse IgG (all from Invitrogen) and horseradish peroxidase (HRP)–conjugated goat anti-mouse IgG and HRP-conjugated donkey anti-rabbit IgG (both from Jackson ImmunoResearch). Reagents were Hanks' balanced salt solution (HBSS; Invitrogen), protease inhibitor cocktail (Roche), IgG-free bovine serum albumin (BSA) (Jackson ImmunoResearch), and DAF-FM diacetate, Fluo-4 AM, and eosin-5-maleimide (all from Invitrogen).

RBC preparation and deposition of complement fragments.

Blood was obtained from healthy adult volunteers in accordance with the guidelines of the Institutional Review Board of Beth Israel Deaconess Medical Center and after informed consent was obtained in accordance with the Declaration of Helsinki. RBCs (∼50 μl) from healthy universal donors (type O, Rh negative) were obtained by finger prick, resuspended in 1 ml of HBSS with Ca++ and Mg++ (HBSS++; Invitrogen), and washed twice before use. RBCs were then resuspended in HBSS++ with 0.5% IgG-free BSA at a hematocrit value of 20%. RBCs were incubated with 20% serum from either SLE patients or normal donors (matched for age, sex, and race to the SLE patient) for 15 minutes at 37°C, and then washed twice and resuspended in HBSS++.

Serum depletion of C4.

One milliliter of control or SLE sera was incubated with 100 μl of protein A–Sepharose beads (Pierce) coupled to either IgG control or anti-C4 mAb for 30 minutes at 4°C. To prevent complement activation due to immobilized IgG, EDTA was added to the sera to a final concentration of 5 mM for 10 minutes prior to immunodepletion. At the completion of the reaction, Sepharose beads were removed by centrifugation, and initial serum concentrations of Ca++ (1.5 mM) and Mg++ (1 mM) were restored.

Immunofluorescence microscopy.

RBCs isolated from healthy universal donors were incubated for 30 minutes at 37°C with 20% sera from healthy donors or SLE patients diluted in HBSS++. Cells were then washed twice and incubated with 10 μg/ml anti-C4c mAb for 10 minutes in HBSS++ with 0.5% BSA (staining buffer), washed, and stained with Alexa Fluor 594–conjugated goat anti-mouse IgG for an additional 15 minutes. After staining, cells were washed twice, mounted in fluorescence mounting media (DakoCytomation), and imaged using an Olympus BX62 microscope fitted with a cooled Hamamatsu Orca AG camera. The microscope, filters, and camera were controlled using iVision version 4.0.9 software (BioVision).

Measurements of RBC membrane flickering.

Phase-contrast time-lapse images of RBCs seeded on microscope slides were recorded for 10 seconds, representing 250 frames, using a 100× UPlanApo phase-contrast objective on an Olympus BX62 microscope. Changes in intensity due to oscillation of RBC membranes (flickering) were measured using iVision version 4.0.9 software. At the end of each recording, an intensity projection step of the image stack was included to identify and exclude RBCs that had drifted during recording.

Flow cytometry.

Fresh or serum-exposed RBCs were incubated for 15 minutes with antibodies in staining buffer at 4°C, followed by 2 washes and incubation for 15 minutes with Alexa Fluor 488–conjugated secondary antibodies specific for each primary antibody at a dilution recommended by the manufacturer. After incubation with secondary antibodies, RBCs were washed once and analyzed using an LSRII flow cytometer (BD Biosciences). In all experiments, at least 10,000 events were recorded and analyzed (FlowJo version 9.0.1 software; Tree Star).

Eosin-5-maleimide labeling.

RBCs were incubated with 25% control or SLE sera for 30 minutes at 37°C. RBCs were washed and incubated in the presence of HBSS++ with eosin-5-maleimide at a final concentration of 0.1 mg/ml for 20 minutes in the dark at room temperature. They were then washed 4 times with 1,000 μl of HBSS++ and analyzed by flow cytometry as described above.

Analysis of RBC calcium influx.

RBCs (∼108) were preloaded with Fluo-4 AM for 15 minutes at room temperature, washed, and resuspended in HBSS++. They were allowed to sit at room temperature for an additional 10 minutes and washed again to remove any uncleaved Fluo-4 AM. Due to the ATP-depleting effect of the acetoxymethyl group, all experiments were performed within 1 hour after Fluo-4 AM loading of RBCs. Fluorescence levels of RBCs were acquired for 15 seconds using an LSRII flow cytometer to establish a baseline for intra-RBC Ca++ concentration. Fifty microliters of control or SLE sera (12.5% of the final volume) was then added to the RBCs, and the fluorescence intensity associated with intra-RBC Ca++ concentration was recorded for an additional 3–5 minutes. Data were exported as forward scatter 3.0 files without the time dimension and analyzed using the kinetic module of FlowJo version 9.0.1 software.

Analysis of RBC NO production.

RBCs were preloaded with DAF-FM diacetate for 30 minutes at room temperature, washed, and resuspended in HBSS++. They were then analyzed according to the same protocol as described above for Ca++ influx measurements. Certain NO detection experiments were run for 40 minutes post–serum challenge.

Western blotting.

RBCs (3 μl) incubated with normal or SLE serum were lysed in 100 μl of 1× reducing–loading buffer (Invitrogen) and boiled for 4 minutes. Samples were run on 10% Bis-Tris Gels (Invitrogen), transferred to nitrocellulose paper (Pierce), and blocked with 6% nonfat dry milk (Bio-Rad) in Tris buffer with 0.1% Tween for 1 hour at room temperature. Membranes were then incubated with antibodies for 30 minutes at room temperature, washed, and incubated with appropriate HRP-conjugated secondary antibodies for an additional 30 minutes. Nitrocellulose membranes were washed extensively in Tris buffer with 0.1% Tween and developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and imaged using the LAS 4000 imaging system (Fujifilm). The intensity profiles of the protein bands were analyzed using Quantity One version 4.5.2 software (Bio-Rad) and plotted using GraphPad version 4.0 software.

Fabrication of the 2-dimensional (2-D) filters.

To measure the ability of RBCs to undergo capillary-like deformations, we fabricated a pseudo–2-D analog of the 5-μm Nucleopore filter comprising an array of posts with 5-μm openings (channels) between them. The design and fabrication of this microchannel and configuration of the experimental set-up have been described previously in detail (11). Briefly, we patterned a silicon wafer with an image of the 2-D filter in sunken-relief using a direct laser writer (Heidelberg DWL 66; Heidelberg Instruments Mikrotechnik) and reactive ion etching (Bosch process, Unaxis SLR 770 ICP Deep Silicon Etcher; Unaxis USA) at the Cornell University NanoScale Science and Technology Facility. Immediately following assembly, the devices were filled with 1% solution of IgG-free BSA in phosphate buffered saline containing 0.01% sodium azide. We found that devices fabricated in this way can be stored at 4°C and at 100% humidity for at least 6 weeks without any measurable loss of functionality.

Measuring RBC deformability using 2-D filters.

To establish the flow of RBCs through the 2-D filter device, the outlet of the 2-D filter device was connected to a waste reservoir (60-ml syringe; BD Biosciences) with a 60-cm–long piece of PE-60 tubing (Instech Laboratories) filled with HBSS++. The difference between the level of liquid in the inlet of the 2-D filter device and the level of liquid in the waste reservoir provided the driving pressure difference. The zero pressure difference corresponded to the absence of movement of RBCs within the device. RBCs (8 μl, hematocrit 20%) were loaded into the inlet reservoir and allowed to enter the network (for ∼1 minute) by lowering the waste reservoir tubing. Once RBCs entered the capillary area, the waste reservoir tubing was reconnected to the waste reservoir at a height that allowed RBCs to pass through the 25-μm length of the capillary in ∼3 seconds. The passage of RBCs through the 2-D network was recorded using a 40×0.75 Ph2 Plan Fluorite objective on a TE300 Nikon inverted microscope, with a Retiga Exi (QImaging) CCD camera controlled with iVision software at a rate of 10 frames/second. The time-stacks files were analyzed frame by frame, and the results are expressed as the number of seconds necessary for an RBC to pass through a microchannel.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Sera from SLE patients deposit C4d complement fragments on normal RBCs.

In order to evaluate the effect of complement deposition on RBC functions, we incubated normal RBCs from universal donors (type O, Rh negative) with sera from 7 healthy controls or 27 SLE patients. Figure 1A shows that incubation of RBCs with SLE sera promoted significant C4d deposition on RBC membranes, in contrast to sera from matched healthy controls (P = 0.016). We then investigated whether the deposition of complement fragments on RBC membranes was due to activation of the classical complement pathway by the immune complexes present in sera from SLE patients. To examine this, we repeated the incubation of RBCs from healthy universal donors with sera from controls or SLE patients in the presence or absence of the Ca++ chelator, EGTA. As shown in Figure 1B, removal of Ca++ by addition of 10 mM EGTA significantly inhibited the SLE serum–promoted deposition of C4d on RBC membranes. Thus, our results suggest that activation of the classical complement pathway, either by immune complexes present in SLE sera or by anti-RBC antibodies, is responsible for the deposition of C4d on RBC membranes.

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Figure 1. Systemic lupus erythematosus (SLE) sera promote C4d deposition on red blood cell (RBC) membranes. RBCs from healthy universal donors (type O, Rh negative) were incubated with sera from healthy volunteers or SLE patients. The expression of C4d on the surface of RBCs was measured by flow cytometry and expressed as mean fluorescence intensity. A, Cumulative results of C4d deposition on RBCs incubated with sera from healthy individuals (n = 7) or SLE patients (n = 27). Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. B, Expression of C4d after incubation of healthy control RBCs with SLE serum (from patient L1) in the absence (top) or presence (bottom) of EGTA. One experiment representative of 3 is shown.

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To discriminate between these two mechanisms, we incubated RBCs with sera from SLE patients and measured the levels of IgG and IgM on their membranes. Our results show that SLE sera did not promote significant IgG deposition on RBC membranes, whereas incubation with 2 of the 5 SLE sera tested led to IgM deposition on RBC membranes (for further details, please contact the corresponding author). We therefore concluded that the observed deposition of C4d on RBC membranes was probably due to activation in solution of complement by circulating immune complexes (12, 13), followed by passive deposition of C4d on bystanding RBCs (14). In certain cases, it is possible that nonhemolytic, complement-fixing, anti-RBC IgM antibodies in SLE patients (15) deposit complement fragments directly on the surface of RBCs. Finally, although in SLE complement is primarily activated through the classical pathway, the lectin pathway may also contribute to the generation of C4 fragments.

SLE serum decreases RBC membrane deformability.

Since deposition of C3 and C4 fragments on RBC membranes leads to a significant decrease in membrane deformability (4), we investigated whether SLE serum–dependent deposition of C4d on normal RBC membranes would induce a similar decrease in membrane deformability. RBCs from universal donors were exposed to control sera or sera from SLE patients with mild-to-moderate disease activity. We then evaluated RBC membrane deformability by using a microfluidic device (11, 16) (Figure 2A) and measuring the time necessary for RBCs to pass through a microchannel. As a positive control for complement-mediated decreased RBC membrane deformability, we deposited complement fragments on RBC membranes by activating the alternative complement pathway using cobra venom factor and C7-deficient serum, as previously described (4) (Figure 2B). Our results (Figures 2C and D) show that incubation with SLE sera significantly delayed the passage time of RBCs through the microchannels compared with incubation with control sera (mean ± SD 26.56 ± 20.83 seconds versus 12.65 ± 4.35 seconds; P < 0.001). It has to be noted that there was significant heterogeneity in the effect of SLE serum on individual RBCs. In addition, there was no clear correlation between patient SLEDAI score or specific disease manifestations and the extent of loss of RBC membrane deformability induced by SLE serum.

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Figure 2. SLE sera decrease RBC membrane deformability. A, Shown are serial snapshots of an RBC passing through a 2-dimensional (2-D) filter. B, RBCs were incubated with C7-depleted serum and cobra venom factor in the presence (control RBCs) or absence (complement-decorated RBCs) of EGTA. The RBC deformability was then analyzed using 2-D filters (see Patients and Methods). C, RBCs from healthy universal donors were incubated with sera from healthy individuals (N1 and N2) or SLE patients (L1–L3). RBC deformability was measured using 2-D filters. Each circle represents 1 RBC. Horizontal lines indicate the mean. D, Shown are cumulative results from 4 control and 10 SLE sera. Data in B and D are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. See Figure 1 for other definitions.

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Next, we investigated whether circulating RBCs from SLE patients also displayed the same phenotype. We analyzed 3 patients with active SLE (as defined by a SLEDAI score of >4), 2 patients with inactive disease (as defined by a SLEDAI score of <4), and 9 healthy controls. All patients with active disease showed significant decrease in membrane deformability expressed as increased passage time (mean ± SD 17.86 ± 3.01 seconds), compared with normal controls (mean ± SD 10.11 ± 1.59 seconds) or patients with inactive disease (mean ± SD 11.325 ± 0.46 seconds) (P = 0.001).

Complement deposition inhibits RBC membrane flickering.

Besides membrane deformability, the ability of RBCs to pass through capillaries is also affected by RBC membrane flickering (17), a continuous oscillation of the RBC membranes at amplitudes varying between 20 and 400 nm and at frequencies between 0.2 and 20 Hz (18). We analyzed RBC membrane flickering by measuring changes in light scattering at the surface of RBCs using phase-contrast time-lapse microscopy at a rate of 20 frames/second, as previously described (19). RBCs from universal donors were incubated with sera from 2 controls or 3 SLE patients, and the extent of C4d deposition on RBC membranes was quantified before time-lapse recording of membrane flickering by fluorescence microscopy. As shown in Figure 3, we found an inverse correlation between the amount of C4d deposition on RBC membranes and the amplitude of membrane flickering. Changes in flickering amplitude were expressed as the SD of flickering values. There was a 10–80% decrease in flickering amplitude of 40 analyzed SLE serum–treated RBCs compared with that of 35 analyzed control serum–treated RBCs. Cells that showed an almost complete inhibition of membrane flickering (Figure 3C) represented ∼5% of the total RBC population (Figure 3D). Most of the RBCs displayed intermediate C4d staining (Figure 3B) that resulted in 10–45% inhibition of flickering amplitude. Our results suggest that, besides affecting the deformability of RBC membranes, SLE serum–induced complement deposition is associated with a significant decrease in RBC membrane flickering.

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Figure 3. Sera from SLE patients inhibit RBC membrane flickering. RBCs from healthy universal donors were incubated with serum from SLE patients and stained for C4d deposition as described in Patients and Methods. A–C, Inverse correlation between the amplitude of membrane flickering (A–C) and the amount of C4d deposition on RBC membranes (insets in A–C). Original magnification × 1,000. Shown are amplitudes of membrane flickering of RBCs displaying no C4d deposition (A), moderate C4d deposition (B), and high C4d deposition (C). Results are representative of those from 3 independent experiments. D, Cumulative results from 35 RBCs incubated with sera from 2 controls and 40 RBCs incubated with sera from 3 SLE patients. Horizontal lines indicate the mean. See Figure 1 for definitions.

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SLE serum promotes RBC Ca++ influx and changes in phosphorylation levels of β-spectrin and band 3.

RBC membrane deformability depends on the Ca++-dependent phosphorylation status of cytoskeletal proteins such as β-spectrin and band 3 (20–22). We therefore sought to determine whether serum from SLE patients induces a Ca++ influx in RBCs that would initiate the events responsible for the observed decrease in RBC membrane deformability. RBCs from healthy universal donors were preloaded with Fluo-4 AM for 15 minutes, washed, and incubated with sera from either healthy controls or SLE patients, and changes in the intra-RBC Ca++ levels were determined by time-lapse flow cytometry. Figures 4A and B show that sera from SLE patients triggered a sustained (>30 minutes) RBC Ca++ influx, whereas sera from healthy controls did not.

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Figure 4. Sera from SLE patients trigger Ca++ influx and change the phosphorylation profile of β-spectrin and band 3 in RBCs from healthy donors. A, RBCs from healthy universal donors were preloaded with Fluo-4 AM and incubated with serum from a healthy control (N) or from SLE patients (L2 and L5), and RBC Ca++ influx was measured by flow cytometry. B, Shown is a graph of Ca++ influx triggered by SLE serum. The mean value of the mean fluorescence intensity associated with Ca++ concentration during the entire recording was used as representative fluorescence for each sample. Horizontal lines indicate the mean. C, SLE serum induces dephosphorylation of β-spectrin. RBCs from healthy universal donors were incubated with serum from 1 healthy control (N) or 6 SLE patients (L1–L6) for 15 minutes at 37°C. RBCs were then lysed, and the phosphorylation levels of β-spectrin were detected using antiphosphoserine/threonine monoclonal antibodies. Lower panel represents actin loading control of RBC samples. D, Shown are ratios of the level of phosphorylated β-spectrin to the level of actin in C. E, SLE serum induces phosphorylation of band 3. Band 3 phosphorylation in RBCs was measured after staining RBCs with eosin-5-maleimide as described in Patients and Methods. RBCs incubated with normal serum (N2) or SLE sera (L1 and L2) were stained with eosin-5-maleimide and analyzed by flow cytometry. Autofluorescence of unlabeled RBCs is shown as a solid histogram. F, Shown are cumulative results of band 3 phosphorylation in RBCs incubated with control (n = 7) or SLE (n = 23) sera. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. See Figure 1 for other definitions.

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The RBC cytoskeleton consists of elongated α- and β-spectrin tetramers associated with short actin filaments and accessory proteins that form a dense elastic network on the cytoplasmic side of the plasma membrane (for review, see ref.23). A decrease in the levels of serine phosphorylation of β-spectrin was shown to correlate positively with reduced RBC membrane deformability (20). Therefore, based on our finding regarding the effect of SLE serum on RBC membrane deformability, we hypothesized that SLE serum promotes the dephosphorylation of β-spectrin. RBCs from healthy universal donors were incubated with control or SLE sera and then lysed, and the phosphorylation levels of β-spectrin were measured by Western blotting using antiphosphoserine mAb. Our results (Figures 4C and D) show that, compared with sera from healthy donors, sera from SLE patients decreased the serine phosphorylation levels of β-spectrin, thus providing a mechanistic explanation for the decreased membrane deformability induced by SLE sera and measured by 2-D microchannel array (Figure 2).

Conversely, decreased RBC membrane deformability was recently shown to be associated with increased levels of band 3 tyrosine phosphorylation (24). Therefore, we tested the effect of SLE sera on the tyrosine phosphorylation levels of RBC band 3 by flow cytometry using eosin-5-maleimide. The binding of eosin-5-maleimide to lysine 430 on the extracellular loop of band 3 parallels tyrosine phosphorylation levels of band 3 through a mechanism that is currently poorly understood (24). In accordance with deformability data and β-spectrin phosphorylation results, incubation of RBCs from normal universal donors with SLE sera (n = 23) significantly (P = 0.035) increased the tyrosine phosphorylation of RBC band 3 compared with healthy control sera (n = 7) (Figures 4E and F).

To further confirm that the effect of SLE serum on RBC membranes is due to complement deposition, we depleted the serum from an SLE patient of C4 by immunodepletion and measured its biologic efficacy in inducing band 3 phosphorylation. Our results show that depletion of SLE sera of C4 (Figure 5A) resulted in a diminished deposition of C4 fragments on RBC membrane compared with the original SLE sera and also resulted in modest phosphorylation of band 3 (Figure 5B).

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Figure 5. Depletion of C4 from SLE serum prevents increase in RBC band 3 phosphorylation. Serum from SLE patient L1 was incubated with either IgG control (L1 serum) or anti-C4c monoclonal antibodies coupled to Sepharose beads (C4-depleted serum) in the absence of Ca++ and Mg++ for 30 minutes. RBCs were then incubated with either the control or the C4-depleted serum, and RBC C4d expression levels (A) and band 3 phosphorylation (B) were measured by flow cytometry as described in Patients and Methods. Autofluorescence of unlabeled RBCs is shown as a solid histogram. See Figure 1 for definitions.

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Overall, these results suggest that the SLE serum–induced decrease in RBC deformability is mediated by altered Ca++-dependent phosphorylation of cytoskeletal proteins.

SLE serum induces the production of NO by RBCs.

RBCs express functional endothelial cell NO synthase (eNOS, NOS3), which allows them to generate biologically active NO (25). RBC eNOS is activated by shear stress–generated Ca++ influx, L-arginine, and phosphorylation via phosphatidylinositol 3-kinase (26, 27). We therefore hypothesized that Ca++ influx triggered by sera from SLE patients would increase RBC production of NO. RBCs from normal universal donors were preloaded with DAF-FM diacetate and incubated with sera from healthy controls or SLE patients. As shown in Figures 6A and B, sera from SLE patients promoted a significant increase (P = 0.028) in intra-RBC production (percent over control ± SD 151.4 ± 47.53) of NO compared with sera from healthy individuals (percent over control ± SD 104.8 ± 4.42), suggesting that in SLE, complement-decorated RBCs could represent a significant source of intravascular NO.

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Figure 6. SLE serum induces the production of nitric oxide (NO) by RBCs. A, RBCs preloaded with DAF-FM diacetate were incubated with serum from a normal individual (N2) or from SLE patients (L1 and L3) for 15 minutes at 37°C. RBC autofluorescence is shown as a solid histogram. B, Cumulative data on RBC NO production generated with sera from 4 controls and 8 SLE patients. Horizontal lines indicate the mean. See Figure 1 for other definitions.

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In conclusion, we present evidence for the first time that in SLE, complement fragment deposition on RBC membrane alters the phosphorylation pattern of spectrin and band 3, decreases membrane deformability and flickering, and promotes RBC production of NO.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

SLE is characterized by ongoing activation of complement followed by deposition of complement fragments on circulating RBCs (28). Here we present evidence that ex vivo deposition of C4d on RBCs significantly decreases RBC membrane deformability and flickering, thus negatively affecting the ability of RBCs to flow through capillary-like microchannels. The underlying mechanism for this phenomenon is SLE serum–induced Ca++ influx that changes the phosphorylation status of the key cytoskeletal proteins β-spectrin and band 3. In addition, SLE serum–incubated RBCs generate NO in significant amounts.

The complete range of functional repercussions of the observed alterations in RBC deformability by SLE serum remains to be determined. Diminished RBC membrane deformability may result in decreased ability of RBCs to deliver O2 in certain tissues with narrow capillaries (4–6 μm in diameter) such as the brain and muscle (29, 30). Our observation that SLE RBCs have decreased membrane deformability may, together with chronic anemia, explain some of the constitutional symptoms of patients with SLE such as chronic fatigue (31) and cognitive dysfunction (32, 33).

Another important observation in this study is that complement-decorated RBCs produce significant amounts of NO. RBC-generated NO has been recently recognized as a key signaling molecule, directly affecting vascular endothelial cells and smooth muscle cells under physiologic conditions (34). Moreover, NO was also identified as a significant factor in promoting signaling abnormalities in SLE T cells and in inducing mitochondrial proliferation and hyperpolarization (35, 36). Mechanistically, extracellular NO and its derivatives, such as peroxynitrate, have been directly implicated in the pathogenesis of SLE by modifying the functions of key enzymes and altering the immunogenicity of self antigens (for review, see ref.37). Thus, our findings regarding complement-induced RBC NO production strongly suggest that RBCs may play a significant role in SLE pathophysiology.

In conclusion, we describe the effect of SLE serum on RBC membrane deformability and NO production. The altered RBC functional properties that we observed could explain several of the systemic and local manifestations of SLE. Therefore, RBCs may prove to be not only a good SLE biomarker but also an important therapeutic target for intercepting abnormal signaling events that characterize SLE immunopathology.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Kyttaris had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Ghiran, Zeidel, Shevkoplyas, Tsokos, Kyttaris.

Acquisition of data. Ghiran, Shevkoplyas, Burns, Kyttaris.

Analysis and interpretation of data. Ghiran, Shevkoplyas, Kyttaris.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to thank Dr. Madhukar Shinde, Joseph Khoory, and Ann Mary Philip for their technical assistance with Western blotting analysis, flow cytometry, and microchannel deformability studies.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES