Cell-derived microparticles : composition and function - Chapter 11: Complement activation on cell-derived microparticles in myocardial infarction is mediated by immunoglobulin G and not C-reactive protein

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Cell-derived microparticles : composition and function

Biró, É.

Publication date

2008

Link to publication

Citation for published version (APA):

Biró, É. (2008). Cell-derived microparticles : composition and function.

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HAPTER

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Complement activation on cell-derived

microparticles in myocardial infarction

is mediated by immunoglobulin G

and not C-reactive protein

P. Marc van der Zee

_{, Éva Biró}

_{, Yung Ko}

_{, Robbert J. de Winter}

_{, C.}

Erik Hack

_{, Augueste Sturk}

_{, Rienk Nieuwland}

Submitted.

Abstract

Background: C-reactive protein (CRP), when bound to membranes such as cell-derived

microparticles, activates complement. Recent studies suggested the involvement of microparticles in complement activation and in myocardial infarction. Also, complement activation contributes to the development of atherosclerosis and its complications, including myocardial infarction (MI).

Objectives: Here, we studied complement activation on circulating microparticles in MI

patients and healthy controls.

Methods: Microparticles isolated from plasma of MI patients (n = 21) and sex- and

age-matched healthy individuals (n = 10) were analyzed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (CRP, serum amyloid P component, immunoglobulins IgM and IgG). Concurrently, fluid phase complement activation products and activator molecules were determined.

Results: In MI patients, but not in healthy individuals, microparticles with bound IgG were

strongly associated with C1q exposing microparticles (r = 0.73, P < 0.001), which in turn were strongly associated with microparticles exposing C4 and those exposing C3 (r = 0.78 and r = 0.87, respectively; both P < 0.001). In contrast, in healthy individuals microparticles with bound CRP were strongly associated with those exposing C1q (r = 0.82, P = 0.007), which in turn were associated with those exposing C4 and those exposing C3 (r = 0.68, P = 0.032 and r = 0.68, P = 0.031, respectively). Fluid phase CRP, microparticles with bound CRP, and C3 activation products were elevated in MI patients (mean 20.90 versus 1.66 mg/L, P = 0.032, 463 × 106_{/L versus 197 × 10}6_{/L, P = 0.031, and}

28.07 versus 20.34 nmol/L, P = 0.023 respectively), and fluid phase CRP correlated with microparticles with bound CRP (r = 0.84, P < 0.001).

Conclusions: Despite CRP-associated complement activation on the surface of

microparticles in healthy individuals and a strong correlation between microparticle-bound CRP and fluid phase CRP in MI patients, microparticle-associated complement activation is IgG- but not CRP-associated in MI patients.

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nflammation plays a crucial role in the development of atherosclerosis and its complications. The pathways of this process, however, remain largely unknown [1]. As elements of the immune system, elevated systemic levels of complement component C3 [2] and activators of the complement system, C-reactive protein (CRP) [3], serum amyloid P component (SAP) [4] and IgG [5] predict adverse future cardiovascular events. In the time course of myocardial infarction (MI), CRP becomes deposited in the infarcted area and contributes to myocardial damage by promoting complement activation [6,7], thereby aggravating ischemia/reperfusion injury [8,9]. In rats, human CRP enhances MI size by activating complement [10]. Moreover, the development of acute cardiac failure has been reported to be more closely associated with the magnitude of the acute phase reaction than with infarction size as measured by CKMB [11].

Previously, we found that in rheumatoid arthritis patients and healthy individuals levels of circulating cell-derived microparticles with CRP on their surface correlated with levels of circulating microparticles with C1q. In turn, these CRP exposing microparticles correlated with microparticles exposing C4, suggesting in vivo classical pathway activation by microparticle-exosed CRP [12]. Microparticles are small vesicles released from cells upon activation or apoptosis exposing negatively charged phospholipids. These provide a surface for the binding of CRP, SAP and immunoglobulins. Once bound, CRP [13-16], SAP [17,18], IgG [12,19] and IgM [20,21] activate the complement system by binding of C1q. Both binding of C1q, as well as deposition of downstream complement components C4 and C3 can occur on microparticles [22,23]. Hence, it can be postulated that microparticles may play an important role in MI by mediating CRP-induced complement activation.

In the present study we studied the pathway of complement activation on the surface of circulating microparticles from MI patients as well as healthy controls. Levels of circulating microparticles with bound C1q and activated C4 and/or C3 were assessed, as well as levels of microparticles binding the complement activator molecules CRP, SAP, IgM and IgG.

Methods

Patient and healthy subject groups

This investigation conforms with the principles outlined in the Declaration of Helsinki. Blood samples were obtained from patients with ST-elevation MI (STEMI; n = 10) and patients with non-ST-elevation MI (NSTEMI; n = 11) [24] at the Department of Cardiology (Academic Medical Center, Amsterdam, the Netherlands) before undergoing catheterization and/or percutaneous coronary intervention. All NSTEMI patients were sampled within 12 hours, and all STEMI patients within 6 hours after onset of symptoms. All patients received medication according to standard clinical practice. None received

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coumarin derivatives, thrombolytic therapy, or anti-platelet medication other than aspirin. All patients received aspirin and LMWH upon admission.

Blood samples were also obtained from healthy individuals, age- and sex-matched to the patient groups (n = 10), as well as from healthy individuals less than 50 years of age (n = 10).

Blood sample collection

Venous blood was collected from the cubital vein into 1/10th _{volume of 105 mmol/L}

trisodium citrate (BD, San José, CA, USA) using minimal venous occlusion. Within 30 min, blood samples were centrifuged (20 min at 1550 × g) at room temperature. Only the upper 2/3 of the apparent plasma fraction were collected. Aliquots of 250 μL were immediately snap frozen in liquid nitrogen for at least 15 min, to be finally stored at −80°C until assay.

Measurement of fluid phase complement activation products and complement activator molecules

Plasma samples (250 μL aliquots) were thawed on melting ice and freed from microparticles by centrifugation at 19,000 × g for 60 min at 4°C. The upper 200 μL of the microparticle-free supernatants were collected and analyzed for concentrations of the soluble complement activation products C4b/c (C4b, inactivated C4b and its further degradation product C4c) and C3b/c (C3b, inactivated C3b and its further degradation product C3c) as well as SAP, as described previously, by enzyme-linked immunosorbent assays [25,26]. CRP, IgM and IgG concentrations were analyzed on the automated clinical chemistry analyzer Modular Analytics P800 using Tina-quant reagents (Roche Diagnostics, Basel, Switzerland).

Flow cytometric analysis of microparticles and bound complement components or complement activator molecules

Microparticles were isolated from plasma as we described previously [27]. Samples (250 μL aliquots) were thawed on melting ice, then centrifuged at 19,000 × g for 30 min at room temperature to pellet the microparticles. Subsequently, 225 μL of the supernatants were removed, and 225 μL of phosphate buffered saline (PBS; 154 mmol/L NaCl, 1.4 mmol/L phosphate, pH 7.4) containing 10.5 mmol/L trisodium citrate (PBS/citrate) were added. Microparticles were resuspended, then again pelleted by centrifugation, after which 225 μL of supernatant were again removed. To the remaining 25 μL microparticle pellet 75 μL of PBS/citrate buffer were added, and the microparticles were resuspended. Flow cytometric analysis was performed using an indirect staining procedure [28]. Microparticles (5 μL aliquots) were incubated for 30 min at room temperature in a final volume of 50 μL of PBS containing 2.5 mmol/L CaCl2 (PBS/Ca, pH 7.4) and unlabeled mouse monoclonal

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antibodies against bound complement factors (C1q, C4, C3) or bound complement activator molecules (CRP, SAP, IgM, IgG), or the respective isotype-matched control antibodies [clones MOPC-31C (IgG1) and G155-178 (IgG2a) from Becton, Dickinson and Company

(BD) Pharmingen, San José, CA, USA]. The monoclonal antibodies against C1q, C4, C3, CRP and SAP (clones C1q-2, C4-4, C3-15, 5G4, and SAP-14, respectively) have been described previously [15,26,29,30]. Antibodies against the heavy chains of IgM and IgG molecules (clones MH15-1 and MH16-1, respectively) were obtained from Sanquin, Amsterdam, the Netherlands. After incubation with the antibodies, the microparticles were washed with 200 μL of PBS/Ca. Then, rabbit anti-mouse F(ab’)2-phycoerythrin [F(ab’)2

-PE; Dako, Glostrup, Denmark; 5 μL] was added, and the mixtures were again incubated for 30 min at room temperature. Subsequently, 400 μL of buffer were added and the microparticles analyzed on a FACSCalibur flow cytometer with CELLQuest 3.1 software [BD Immunocytometry Systems, San José, CA, USA]. Acquisition was performed for 1 min per sample, during which the flow cytometer analyzed approximately 60 μL of the suspension. Forward scatter and side scatter were set at logarithmic gain. To identify marker positive events, thresholds were set based on microparticle samples incubated with similar concentrations of isotype-matched control antibodies. Calculation of the number of microparticles per liter plasma was based on the particle count per unit time, the flow rate of the flow cytometer, and the net dilution during sample preparation of the analyzed microparticle suspension.

Statistical analysis

Data are expressed as mean ± SD. Continuous data were compared using the t test, and dichotomous variables were compared using the χ2 _{test. Correlations were calculated using}

Pearson’s correlation test. P < 0.05 was regarded as statistically significant.

Results

Clinical characteristics

Table 1 shows the clinical characteristics of persons included in the study. Current smoking, familial coronary artery disease, previous angina pectoris, and use of aspirin and beta blockers were more common among MI patients than healthy individuals.

Concentrations of fluid phase complement activation products and complement activators

Fluid phase levels (Table 2) of the complement activator CRP were significantly higher in MI patients when compared to healthy individuals (P = 0.032). There were no differences between groups in fluid phase levels of SAP, IgM and IgG. Concentrations of the fluid

phase complement activation product C3b/c were increased in patients compared to healthy individuals (P = 0.023), while fluid phase C4b/c showed a tendency towards higher levels in MI patients.

Table 1. Clinical characteristics of persons included in the study. Healthy individuals

n = 10

MI patients

n = 21

P

Age (mean ± SD; years) 64 ± 8 63 ± 11 0.888

Male (n) 9 18 0.739 BMI (mean ± SD; kg/m2_{) 23.3 ± 4.5 26.6 ± 4.2 0.057} Risk factors Hypertension 0 6 0.060 Hypercholesterolemia 0 3 0.209 Diabetes Mellitus 0 2 0.313 Current smoking 0 11 0.004

Familial coronary artery disease1 _{0 11 0.004}

History

Angina 0 13 0.001

Chronic heart failure 0 1 0.483

Peripheral arterial disease 0 1 0.483

CVA / TIA 0 0 1.000

MI 0 5 0.092

Percutaneous coronary intervention 0 2 0.313

Medication Aspirin2 _{0 10 0.008} Beta blockers 0 10 0.008 Calcium antagonists 0 4 0.139 Nitrates 0 4 0.139 Statins 0 6 0.060 ACE inhibitors 0 3 0.209

1_{Documented MI or coronary artery disease in parents or siblings before the age of 60.} 2_{Daily use of}

aspirin in previous 7 days. *Compared with older healthy subjects P < 0.05. ***Compared with older healthy subjects P < 0.001.

ACE, angiotensin-converting enzyme; BMI, body mass index; CVA, cerebrovascular accident; MI, myocardial infarction; TIA, transient ischemic attack.

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Table 2. Concentrations of fluid phase complement activator molecules and complement activation

products in plasma of healthy individuals and myocardial infarction patients.

Healthy individuals (n = 10) MI patients (n = 21) P CRP (mg/L) 1.66 ± 1.47 20.90 ± 38.32 0.032 SAP (mg/L) 61.89 ± 18.43 65.38 ± 19.31 0.636 IgM (g/L) 0.58 ± 0.22 0.52 ± 0.37 0.594 IgG (g/L) 9.55 ± 2.41 7.90 ± 2.17 0.085 C4b/c (nmol/L) 4.22 ± 2.35 6.02 ± 5.12 0.198 C3b/c (nmol/L) 20.34 ± 4.02 28.07 ± 13.09 0.023

Data are presented as mean ± SD. Differences were analyzed using the t test (P, two-tailed significance level, considered significant at P < 0.05).

CRP, C-reactive protein; IgG, immunoglobulin G; IgM, immunoglobulin M; SAP, serum amyloid P component.

Table 3. Concentrations of microparticles with bound complement activator molecules and complement

components in plasma of healthy individuals and myocardial infarction patients.

Healthy individuals (n = 10) MI patients (n = 21) P CRP pos. MP (× 106_{/L) 197 ± 98 463 ± 496 0.031} SAP pos. MP (× 106_{/L) 3238 ± 1217 2820 ± 1449 0.416} IgM pos. MP (× 106_{/L) 1128 ± 606 1414 ± 818 0.291} IgG pos. MP (× 106_{/L) 146 ± 98 152 ± 109 0.873} C1q pos. MP (× 106_{/L) 299 ± 233 284 ± 265 0.875} C4 pos. MP (× 106_{/L) 697 ± 351 705 ± 303 0.948} C3 pos. MP (× 106_{/L) 245 ± 211 352 ± 287 0.258}

Data are presented as mean ± SD. Differences were analyzed using the t test (P, two-tailed significance level, considered significant at P < 0.05).

CRP, C-reactive protein; IgG, immunoglobulin G; IgM, immunoglobulin M; MI, myocardial infarction; MP, microparticles; pos., positive; SAP, serum amyloid P component.

Concentrations of microparticles and microparticle-bound complement activation products and complement activators

Concentrations of microparticles with bound CRP were significantly higher in MI patients compared with healthy individuals (P = 0.031). There were no differences between healthy individuals and MI patients with regard to the concentrations of microparticles with bound

SAP, IgM, or IgG. Likewise, concentrations of microparticles with bound complement components (C1q, C4, C3) were similar between the two groups (Table 3).

Correlations between the concentrations of microparticles binding complement activator molecules or complement components

In healthy individuals, concentrations of microparticles with bound CRP correlated strongly with concentrations of microparticles with bound C1q (r = 0.82, P = 0.007, Figure 1A), whereas concentrations of the microparticles with bound complement activators SAP, IgM, or IgG did not (Table 4). In MI patients, concentrations of microparticles with bound CRP correlated with fluid phase CRP levels (r = 0.84, P < 0.001, Table 4), but no correlation was present between microparticles exposing CRP and those exposing C1q (r = 0.32, P = 0.174, Figure 1C). In contrast, in MI patients a strong correlation was present between the concentrations of microparticles with bound C1q and those with bound IgG (r = 0.73, P < 0.001, Figure 1D).

Microparticles with bound C1q did not correlate with those with bound SAP or IgM (Table 4). Levels of microparticles with bound C1q correlated with microparticles with bound C3 and C4 in both healthy individuals and MI patients (Table 4), indicating that in both cases activation of complement occurs via the classical pathway.

Table 4. Correlations between the concentrations of plasma CRP, microparticles binding complement

activator molecules, and microparticles binding complement components in plasma of healthy individuals and myocardial infarction patients.

Healthy individuals (n = 10) MI patients (n = 21) r P r P CRP vs. CRP pos. MP -0.22 0.564 0.84 <0.001 CRP pos. MP vs. C1q pos. MP 0.82 0.007 0.32 0.174

SAP pos. MP vs. C1q pos. MP -0.28 0.441 0.06 0.804

IgM pos. MP vs. C1q pos. MP 0.02 0.964 -0.02 0.946

IgG pos. MP vs. C1q pos. MP 0.56 0.090 0.73 <0.001

C1q pos. MP vs. C4 pos. MP 0.68 0.032 0.78 <0.001

C1q pos. MP vs. C3 pos. MP 0.68 0.031 0.87 <0.001

Pearson’s correlation test was performed (r, correlation coefficient; P, two-tailed significance level, considered significant at P < 0.05).

CRP, C-reactive protein; IgG, immunoglobulin G; IgM, immunoglobulin M; MP, microparticles; pos., positive; SAP, serum amyloid P component.

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IgG positive microparticles (x106/L)

C 1q posi ti ve m icr o p ar ti cl es ( x1 0 6 /L ) 0 500 1000 1500 2000 2500 0 200 400 600 800 1000 1200 r = 0.32 P = 0.174 CRP positive microparticles (x106_/L) C 1q posi ti ve m icr o pa rt icl es ( x1 0 6 /L ) 0 100 200 300 400 0 200 400 600 800 1000 1200 1400 r = 0.73 P < 0.001

IgG positive microparticles (x106_/L)

C 1q posi ti ve m icr o pa rt icl es ( x1 0 6 /L ) A B

Figure 1. Scatter plots of concentrations of microparticles positive for CRP or IgG, versus

concentrations of microparticles positive for C1q in A. healthy individuals and B. MI patients. Pearson’s correlation test was performed (r, correlation coefficient; P, two-tailed significance level, considered significant at P < 0.05).

CRP, C-reactive protein; IgG, immunoglobulin G.

Discussion

Despite studies on the role of CRP-mediated complement activation during MI in animals [10,31], limited human in vivo data exist in this field. Our study shows that during the course of MI in humans, complement activation occurs on circulating microparticles, with increased fluid phase CRP and C3 activation products in plasma. Interestingly, complement activation on microparticles in MI patients was related to microparticle-bound IgG rather than CRP, which was in marked contrast to (matched) healthy controls.

In previous studies, we found differential associations between microparticle-bound CRP and complement activation, depending on pathology. In plasma of rheumatoid arthritis patients, microparticles with bound CRP were implicated in complement activation [12], while in preeclampsia patients [32], as well as in the MI patients reported in the present

study, CRP exposing microparticles were not associated with complement activation on their surface.

The absence of CRP-induced complement activation in MI may reflect protection from complement-induced tissue injury by e.g. complement inhibitors like C4-binding protein (C4-bp), which inhibit CRP-mediated complement activation on membranes [33]. Moreover, both proteins are acute phase reactants [34] , and hence the inhibition mechanism by C4-bp may be relevant to protect host tissues against excessive complement activation during acute phase reactions.

The association in MI patients between IgG exposing microparticles and those exposing bound complement activation products is in line with the predictive value of IgG for future MI [5]. Systemic concentrations of IgG exposing microparticles were comparable between MI patients and healthy individuals in our study. However, IgG binding to microparticles might have different binding specificities in MI patients and healthy individuals, or, alternatively, IgG in patients and healthy individuals might even have a different mode of binding to the microparticles (via Fab fragments and corresponding antigenic epitopes on microparticle surface versus Fc fragments and Fc receptors on the microparticles) with different (patho)physiological sequelae.

In conclusion, our results suggest that complement activation on microparticles in MI occurs via a different molecular mechanism than that in healthy conditions. This may have pathogenic consequences, and if so, may also be a future therapeutic target.

Acknowledgements

Funding for this work was provided by the Netherlands Heart Foundation (grant no. 2000B136).

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