C4d deposits on the surface of RBCs in trauma patients and interferes with their function

Abstract

Objective: Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on RBCs, we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function.

Design: A prospective experimental study.

Setting: Research laboratory.

Subjects: Blood samples collected from 42 trauma patients and 21 healthy donors.

Intervention: None.

Measurements and main results: RBC and sera were collected from trauma patients and control donors. RBCs from trauma patients (n = 40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared with RBCs from control (n = 17) (p < 0.01). Increased amounts of iC3b were found in trauma sera (n = 27) (vs 12 controls, p < 0.01) by enzyme-linked immunosorbent assay. Incubation of RBC from universal donors (type O, Rh negative) with trauma sera (n = 10) promoted C4d deposition on their surface (vs six controls, p< 0.05). Complement-decorated RBC (n = 6) displayed limited their deformability (vs six controls, p < 0.05) in two-dimensional microchannel arrays. Incubation of RBC with trauma sera (n = 10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs eight controls, p < 0.05), and also accelerated calcium influx (n = 9) and enhanced nitric oxide production (n = 12) (vs four and eight controls respectively, p < 0.05) in flow cytometry.

Conclusions: Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunctions, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms.

Figure 1. Increased C4d deposition on the surface of membrane of red blood cells from trauma

A, Deposition of C4d on the surface of red blood cells from trauma patients and healthy donors was measured by flow cytometry. Representative of experiments is shown. B, Data of C4d deposition on red blood cells from all samples was expressed as mean fluorescence intensity and cumulative data from trauma patients (n=40) or normal healthy donors (n=17) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Serum levels of iC3b fragments from trauma patients (n=27) and from normal healthy donors (n=12) were assayed as a measure of activation of complement. Horizontal lines indicate the median.

Figure 1. Increased C4d deposition on the surface of membrane of red blood cells from trauma

A, Deposition of C4d on the surface of red blood cells from trauma patients and healthy donors was measured by flow cytometry. Representative of experiments is shown. B, Data of C4d deposition on red blood cells from all samples was expressed as mean fluorescence intensity and cumulative data from trauma patients (n=40) or normal healthy donors (n=17) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Serum levels of iC3b fragments from trauma patients (n=27) and from normal healthy donors (n=12) were assayed as a measure of activation of complement. Horizontal lines indicate the median.

Figure 1. Increased C4d deposition on the surface of membrane of red blood cells from trauma

A, Deposition of C4d on the surface of red blood cells from trauma patients and healthy donors was measured by flow cytometry. Representative of experiments is shown. B, Data of C4d deposition on red blood cells from all samples was expressed as mean fluorescence intensity and cumulative data from trauma patients (n=40) or normal healthy donors (n=17) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Serum levels of iC3b fragments from trauma patients (n=27) and from normal healthy donors (n=12) were assayed as a measure of activation of complement. Horizontal lines indicate the median.

Figure 2. Increased C4d deposition on healthy red blood cell membranes incubated with trauma sera

A. Deposition of C4d on the surface of healthy red blood cells (type O, Rh negative) incubated with sera from trauma patients or healthy donors were measured by flow cytometry. Representative data is shown. B. Data of C4d deposition on red blood cells from all samples was expressed as mean fluorescence intensity, and cumulative data from trauma patients (n=10) or healthy donors (n=6) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Deposition of C4d on the surface of healthy red blood cells (type O, Rh negative) incubated with sera from trauma patients with or without 10 mM EDTA. Representative data is shown.

Figure 2. Increased C4d deposition on healthy red blood cell membranes incubated with trauma sera

A. Deposition of C4d on the surface of healthy red blood cells (type O, Rh negative) incubated with sera from trauma patients or healthy donors were measured by flow cytometry. Representative data is shown. B. Data of C4d deposition on red blood cells from all samples was expressed as mean fluorescence intensity, and cumulative data from trauma patients (n=10) or healthy donors (n=6) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Deposition of C4d on the surface of healthy red blood cells (type O, Rh negative) incubated with sera from trauma patients with or without 10 mM EDTA. Representative data is shown.

Figure 2. Increased C4d deposition on healthy red blood cell membranes incubated with trauma sera

A. Deposition of C4d on the surface of healthy red blood cells (type O, Rh negative) incubated with sera from trauma patients or healthy donors were measured by flow cytometry. Representative data is shown. B. Data of C4d deposition on red blood cells from all samples was expressed as mean fluorescence intensity, and cumulative data from trauma patients (n=10) or healthy donors (n=6) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Deposition of C4d on the surface of healthy red blood cells (type O, Rh negative) incubated with sera from trauma patients with or without 10 mM EDTA. Representative data is shown.

Figure 3. Decreased red blood cell membrane deformability after incubation with trauma sera

A. A composite series of images illustrating the passage of a single red blood cell through a capillary-size microchannel of the 2- dimensional filter device B. Healthy universal donor's (type O, Rh negative) red blood cells deformability were measured by using the 2-dimensional filter device after incubation with sera from trauma patients or healthy donors. Decreased deformability of each red blood cell was associated with increased transit time (i.e. the time it took the cell to pass through the capillary microchannels of 2-dimensional filter). Data are shown as dot blots of all experiments with each circle showing the passage time for one red blood cell. Horizontal lines show the mean. Representative data are shown. C. Cumulative data from trauma patients (n=6) or healthy donors (n=6) are shown. Data are expressed as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median.

Figure 3. Decreased red blood cell membrane deformability after incubation with trauma sera

A. A composite series of images illustrating the passage of a single red blood cell through a capillary-size microchannel of the 2- dimensional filter device B. Healthy universal donor's (type O, Rh negative) red blood cells deformability were measured by using the 2-dimensional filter device after incubation with sera from trauma patients or healthy donors. Decreased deformability of each red blood cell was associated with increased transit time (i.e. the time it took the cell to pass through the capillary microchannels of 2-dimensional filter). Data are shown as dot blots of all experiments with each circle showing the passage time for one red blood cell. Horizontal lines show the mean. Representative data are shown. C. Cumulative data from trauma patients (n=6) or healthy donors (n=6) are shown. Data are expressed as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median.

Figure 3. Decreased red blood cell membrane deformability after incubation with trauma sera

A. A composite series of images illustrating the passage of a single red blood cell through a capillary-size microchannel of the 2- dimensional filter device B. Healthy universal donor's (type O, Rh negative) red blood cells deformability were measured by using the 2-dimensional filter device after incubation with sera from trauma patients or healthy donors. Decreased deformability of each red blood cell was associated with increased transit time (i.e. the time it took the cell to pass through the capillary microchannels of 2-dimensional filter). Data are shown as dot blots of all experiments with each circle showing the passage time for one red blood cell. Horizontal lines show the mean. Representative data are shown. C. Cumulative data from trauma patients (n=6) or healthy donors (n=6) are shown. Data are expressed as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median.

Figure 4. Phosphorylation status of band 3 in red blood cells incubated with trauma sera

Phosphorylation of band 3 in healthy red blood cells (type O, Rh negative) measured by flow cytometry after incubation with sera from trauma patients or healthy donors, using eosin-5-maleimide staining. Band 3 phosphorylation was expressed as mean fluorescence intensity of eosin-5-maleimide fluorescence, and cumulative data from trauma patients (n=10) or healthy donors (n=8) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median.

Figure 5. Ca²⁺ influx into red blood cell after addition of trauma sera

A, Time course of serum-induced changes in intracellular Ca²⁺ of trauma patients (Tr4 and Tr5) or a healthy control (N3) are shown. Arrow indicates the time when trauma or control sera were added to red blood cell preloaded with Fluo-4 AM, 1 minute after start of measurement by flow cytometry. Vertical axis indicates relative intracellular Ca²⁺ concentration, which is estimated from mean fluorescence intensity of Fluo-4 AM. Representative data are shown. B. Cumulative results of Ca²⁺ influx in red blood cells to which trauma sera (n=9) or normal sera (n=4) were added. Vertical axis shows the ratio of mean fluorescence intensity of Fluo-4 AM before and after adding the serum. Horizontal lines indicate the median.

Figure 5. Ca²⁺ influx into red blood cell after addition of trauma sera

A, Time course of serum-induced changes in intracellular Ca²⁺ of trauma patients (Tr4 and Tr5) or a healthy control (N3) are shown. Arrow indicates the time when trauma or control sera were added to red blood cell preloaded with Fluo-4 AM, 1 minute after start of measurement by flow cytometry. Vertical axis indicates relative intracellular Ca²⁺ concentration, which is estimated from mean fluorescence intensity of Fluo-4 AM. Representative data are shown. B. Cumulative results of Ca²⁺ influx in red blood cells to which trauma sera (n=9) or normal sera (n=4) were added. Vertical axis shows the ratio of mean fluorescence intensity of Fluo-4 AM before and after adding the serum. Horizontal lines indicate the median.

Figure 6. Nitric oxide production from red blood cells induced by trauma serum

A. Nitric oxide production from healthy red blood cells (type O, Rh negative) measured by flow cytometry after incubation with sera from trauma patients or healthy donors by using DAF-FM diacetate. Representative data is shown. B. Nitric oxide production from red blood cells was expressed as mean fluorescence intensity of DAF-FM diacetate fluorescence, and cumulative data from trauma patients (n=12) or healthy donors (n=8) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Heat inactivation of complement in sera from trauma patients eliminates their ability to trigger NO production. To inactivate complement, sera were incubated 55°C for 30 minutes. Red blood cells were incubated with sera from trauma patients before or after inactivation of complement. Individual sera with/without inactivation of complement are shown. Straight lines are used to connect individual points to help visualize how different they are in each sample (n=4).

Figure 6. Nitric oxide production from red blood cells induced by trauma serum

A. Nitric oxide production from healthy red blood cells (type O, Rh negative) measured by flow cytometry after incubation with sera from trauma patients or healthy donors by using DAF-FM diacetate. Representative data is shown. B. Nitric oxide production from red blood cells was expressed as mean fluorescence intensity of DAF-FM diacetate fluorescence, and cumulative data from trauma patients (n=12) or healthy donors (n=8) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Heat inactivation of complement in sera from trauma patients eliminates their ability to trigger NO production. To inactivate complement, sera were incubated 55°C for 30 minutes. Red blood cells were incubated with sera from trauma patients before or after inactivation of complement. Individual sera with/without inactivation of complement are shown. Straight lines are used to connect individual points to help visualize how different they are in each sample (n=4).

Figure 6. Nitric oxide production from red blood cells induced by trauma serum

A. Nitric oxide production from healthy red blood cells (type O, Rh negative) measured by flow cytometry after incubation with sera from trauma patients or healthy donors by using DAF-FM diacetate. Representative data is shown. B. Nitric oxide production from red blood cells was expressed as mean fluorescence intensity of DAF-FM diacetate fluorescence, and cumulative data from trauma patients (n=12) or healthy donors (n=8) are shown as box plots. Each box shows the 25th and 75th percentiles. Lines outside the boxes show the lowest and the highest values. Lines inside the boxes show the median. C. Heat inactivation of complement in sera from trauma patients eliminates their ability to trigger NO production. To inactivate complement, sera were incubated 55°C for 30 minutes. Red blood cells were incubated with sera from trauma patients before or after inactivation of complement. Individual sera with/without inactivation of complement are shown. Straight lines are used to connect individual points to help visualize how different they are in each sample (n=4).