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Abstract

Objectives

Fibronectin (FN) is able to bind fibrin and FN–fibrin complexes and is found in the plasma of some patients suffering from inflammatory disease. The present study was undertaken to determine whether soluble supra-molecular FN–fibrin complexes were present in the plasma of children with recurrent respiratory infections (RRI).

Design and methods

The frequency of occurrence and relative amounts of the supra-molecular FN–fibrin forms, concentrations of immunoglobulins and numbers of natural killer cells (NK) were determined in the plasma of children with recurrent respiratory infections. The frequencies of these parameters were compared with their frequencies in the plasma of children with acute respiratory infections and plasma from healthy children.

Results

SDS-agarose immunoblotting of patients’ plasma revealed the presence of several additional FN–fibrin bands, with decreasing electrophoretic mobilities and increasing molecular masses of 750 kDa, 1000 kDa, 1300 kDa, 1600 kDa and 1900 kDa. Such FN–fibrin complexes occurred with higher frequency and in larger amounts in the plasma of children with RRI and acute infection than they did in plasma from normal children. Moreover, bands above 1000 kDa were absent in most young healthy individuals. The occurrence of FN–fibrin complexes did not correlate with either immunoglobulin concentrations, or with the number of NK cells.

Conclusions

The occurrence of plasma supra-molecular FN–fibrin complexes is associated with acute and recurrent respiratory infections of children.

Keywords

Fibronectin FN–fibrin complexes immunoblotting recurrent respiratory infection NK cells

Introduction

Recurrent respiratory infection (RRI) is one of the most common and frequent diseases in children and adolescents. Its increased incidence in children is mainly related to constitutional (i.e. age of the child, immune system condition, congenital defects of respiratory system, concomitant diseases and allergies) and environmental factors, such as socio-economic conditions, increased exposure to infection, microbial virulence and overuse of antibiotics.^1,2 In patients with RRI, it is more difficult to eliminate pathogens from the body and this can lead to an increased predisposition to recurrent infections. Consequently, RRI in childhood may increase susceptibility to diseases in adulthood, for example, asthma, diabetes, chronic bronchitis and emphysema.³

Fibronectin (FN) is a large glycoprotein, and is present in significant amounts in plasma, cells and the extracellular matrix (ECM). It is involved in a wide variety of cellular functions, including cell adhesion, migration, proliferation and differentiation.⁴ FN, secreted by hepatocytes, circulates in blood at varying concentrations in children,⁵ and is involved in blood clotting, thrombosis, and wound healing processes.^5,6 In the presence of endothelial injury, circulating FN enters the extravascular space and becomes incorporated into the ECM.⁴ The binding of plasma FN to cellular integrin receptors leads to stretching of FN, leading to the formation of a fiber multimeric network.⁷ An insoluble, cellular FN form, produced locally by fibroblasts, macrophages, lymphocytes, platelets and endothelial cells, accumulates locally in tissue,⁴ where it is essential for the assembly and maintenance of the ECM, preserving its proper structural integrity, organization, and regulation of normal tissue metabolism.⁸

The basic FN form is a 440–500 kDa dimer, made of two identical or similar disulphide-bonded polypeptide chains. Both FN polypeptides are composed of three types of repeating I, II and III modules which are arranged into independently folded functional domains with binding sites for ECM proteins (e.g. collagen), cell surface receptors (integrins, bacterial FN receptors), blood protein derivatives (e.g. fibrin) and glycosaminoglycans (e.g. heparin).⁴ Additionally, an alternative splicing of mRNA leads to the inclusion of three extra domains (termed EDA, EDB and IIICS segments) to cellular FN, and only the IIICS segment to plasma FN.^4,8

FN derived from different cell types displays high structural and functional heterogeneity, which may result primarily from alternative splicing of its pre-mRNA and different degrees of post-translational N- and O- glycosylations.⁹ Moreover, FN has an ability to form cross-linked complexes with, for example, FN fragments,¹⁰ various extracellular matrix components,⁸ and some blood plasma components such as fibrin and fibrinogen,⁵ creating high-molecular FN forms. The FN forms with increased molecular masses were shown to be produced by carcinoma cell lines in vitro.^11,12 In a previous study, we have observed the high molecular FN forms, following SDS-PAGE under reducing conditions, in the plasma of patients with neurodegenerative diseases,¹³ and plasma and pleural effusions of some patients with lung cancer and lung inflammation,¹⁴ and also of elderly individuals.⁵ These FN forms were identified as the subunits of soluble supra-molecular plasma FN–fibrin complexes, which were easily revealed by SDS-agarose immunoblotting.¹⁵

The present study was undertaken to determine whether the soluble supra-molecular FN–fibrin complexes occurred in the plasma of children suffering from RRI. The frequency of occurrence and relative amounts of supra-molecular FN–fibrin forms in the plasma of children with RRI, during the period of recovery, stratified by immunoglobulin concentration and number of natural killer (NK) cells were compared to their frequency in those children with acute infection and to their frequency in healthy children.

Materials and methods

Sampling

Blood samples, anti-coagulated with sodium citrate, were collected from 94 children under the medical care of the 3rd Clinic of Pediatrics, Immunology and Rheumatology, of Developmental Age of Wroclaw Medical University (Poland). The samples were collected with the informed consent of parents of children and the study was approved by the local ethics committee (Commission of Bioethics at Wroclaw Medical University, no. KB-445/2012). Plasma was prepared by centrifugation for 15 min at 2000 g and +4 ℃. The samples were stored at −76 ℃ until use. All samples included in the studies consisted of excess plasma remaining after all routine laboratory investigations had been completed.

Children included in the study fulfilled the criteria of recurrent respiratory infections (RRI), i.e. they had six or more respiratory infections per annum, one or more respiratory infections per month involving the upper airways from September to April and three or more respiratory infections per annum involving the lower airways.^1,2 Samples were collected during the recovery period, when they had been symptom-free for at least 3–4 weeks. Individuals with allergy, autoimmune diseases and other chronic diseases and receiving anti-inflammatory drugs were excluded from the study. Patients were stratified into three groups according to concentrations of immunoglobulins A, G and M and number of circulating NK cells: Group 1, RRI-1, had normal concentrations of immunoglobulins and number of NK cells; Group 2, RRI-2, had low concentrations of one or more immunoglobulin and normal numbers of NK cells; and Group 3, RRI-3, had normal immunoglobulin concentrations and low numbers of circulating NK cells. The characteristics of patients are summarized in Table 1. Two more groups were included: Group 4, infection, consisted of children (1 female, 12 males) who did not meet RRI criteria but had infection of the respiratory tract (laryngitis and/or pneumonia and/or bronchitis and/or pharyngitis); Group 5, normal, comprised generally healthy children (8 females and 9 males) without signs of infection and who did not meet the conditions to include them in any of the RRI groups (Groups 1 to 3), but who visited the doctor because of events such as syncope or abdominal pain. They had normal values of routine laboratory blood parameters, normal concentrations of IgG, IgA, IgM and normal numbers of NK cells.

Table 1.

Characteristics of the groups of children with recurrent respiratory infection and blood parameter findings.

Analysed parameters	Groups
Analysed parameters	RRI-1 n = 20	RRI-2 n = 23	RRI-3 n = 21
Female/male age, years	9/11 9.2 ± 4	7/16 6.0 ± 4	8/13 7.4 ± 5
IgG g/l	9.41 8.13–11.35 n = 19	5.78 4.68–6.61 n = 17 9↓	8.45 7.07–10.17 n = 21 1↑
IgA g/l	1.088 1.01–1.56 n = 19	0.175 6.0–34.0 n = 14 7↓	0.969 0.564–1.23 n = 21
IgM g/l	0.973 0.736–0.1273 n = 16	0.54 0.32–1.25 n = 10 3↓	0.917 0.761–1.383 n = 21 1↓
NK-cells cells/µl	201.0 150.0–213.0 n = 11	153.0 109.0–430.0 n = 3	61.0 44.0–83.0 n = 21 21↓
CRP mg/l	0.1 0.01–0.94 n = 20	0.5 0.1–7.1 n = 23 6↑	0.3 0.1–0.6 n = 21 1↑
WBC × 10⁹/l	6.2 5.8–6.6 n = 17	7.6 5.8–10.8 n = 15 1↑	6.4 5.6–7.8 n = 14
Neutophils (%)	43.8 35.4–53.6 n = 17 5↓	39.4 34.0–54.0 n = 14 2↓ 1↑	42.6 33.5–51.9 n = 14 1↓
PLT × 10⁹/l	282.0 236.0–310.0 n = 17	329.0 272–384 n = 16 1↑	256.0 188.0–287.0 n = 14 1↓

Note: RRI-1: normal concentrations of immunoglobulins and normal number of NK cells; RRI-2: decreased concentrations of immunoglobulins and normal number of NK cells; RRI-3: normal concentrations of immunoglobulins and low numbers of NK cells. The values of normal blood parameters are given as median and 25th–75th percentiles. All parameters were determined using standard laboratory techniques and equipment (Beckman Coulter, Inc., Fullerton, USA) apart from CRP (Abbott, USA). WBC: white blood cells; Neutrophils %: neutrophils percentage of white blood cells; PTL: platelets. ↓↑ the number of samples with low (↓) or high (↑) value.

FN concentration

FN concentration was determined by an enzyme-linked immunosorbent assay (ELISA) using a well-defined domain-specific monoclonal antibody directed to a centrally located cell-binding domain of FN (FN30-8; M010 TaKaRa Shuzo Co. Ltd., Shiga, Japan) as described earlier.¹⁶

Western immunoblotting

Plasma samples containing 300 ng of FN were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and immunoblots prepared as described earlier.¹⁶ The immunoblots were scanned and protein band patterns were analysed by densitometry using GelScan V6.0 (BioSciTec GmbH, Frankfurt/Main, Germany).

SDS-agarose FN immunoblotting

Supramolecular forms of plasma FN were revealed by SDS-agarose immunoblotting as described previously.¹⁵

Statistics

Data are presented as means ± standard deviations (SD). Comparisons between groups were performed by means of the Kruskal–Wallis and post hoc tests. P < 0.05 was regarded as significant.

Results

FN reduced bands

Electrophoresis followed by immunoblotting revealed the presence of a high molecular ∼280 kDa FN reduced band, in addition to the normal FN subunit of ∼240 kDa (Figure 1).

Figure 1.

Representative immunoblotting pattern of plasma FN of children suffering from RRI. SDS-PAGE was done under reducing conditions in 5% gel. The immunoblots were developed with monoclonal antibody directed to cellular binding domain FN (TaKaRa Shuzo Co. Ltd., Japan) as described by Pupek et al.¹⁶ In all cases, 300 ng of FN was applied per lane. The immunoblotting was done for all 94 plasma samples, but only the representative patterns of plasma FN are shown: RRI-1 (lane 1), RRI-2 (lane 2), RRI-3 (lane 3), normal plasma of child (lane 4).

The FN reduced band of ∼280 kDa occurred more frequently in the samples of children with RRI, Groups 1–3, and Group 4, the infection group, than it did in the samples from children in Group 5, the normal group (Table 2).

Table 2.

FN concentration and occurrence of reduced FN form in plasma of children.

Group		Number of samples/ age year	FN (mg/l)	Quantification of FN bands in relation to the immunoblotting pattern shown in Figure 1 Frequency of occurrence Mean value of relative amount ± SD
Group		Number of samples/ age year	FN (mg/l)	240 kDa	280 kDa
1.	RRI-1	20 9.2 ± 4	193.23±105.1	1 88.6 ± 11 (20)	1 7.3 ± 8.1 (20)
2.	RRI-2	23 6 ± 4	175.91 ± 61.5	1 85.2 ± 13.1 (23)	0.83 15.6 ± 10.3 (19)
3.	RRI-3	21 7.4 ± 5	160.29 ± 57.4	1 91.9 ± 8.8 (21)	0.47 14.7 ± 10.7 (10)
4.	Infection	13 2.7 ± 2.3	128.92 ± 41.3	1 92.11 ± 4.3 (13)	0.69 4.91 ± 2.4 (9)
5.	Normal	17 7.2 ± 5	196.06 ± 68	1 98.1 ± 4.4 (17)	0.17 8.8 ± 6.2 (3)

Note: The FN reduced forms (240 kDa and 280 kDa) were detected by Western immunoblotting (Figure 1). RRI groups were stratified as described in legend to Table 1. The frequency of occurrence is the ratio of the number of samples containing the reduced FN form to the total number of samples. The relative amount of the FN molecular form (mean value ± SD) corresponds to the percentage of the total number of pixels found in the electrophoresis path. The numbers in parentheses indicate the number of samples which revealed the reduced FN form.

FN multimers

SDS-agarose immunoblotting revealed the presence of several bands with decreasing electrophoretic mobilities (Figure 2a). The band of 500 kDa was a dimer of FN, whereas the bands of 750 kDa, 1000 kDa, 1300 kDa, 1600 kDa and 1900 kDa recognized by anti-FN monoclonal antibody and anti-fibrinogen polyclonal antibodies (Figure 2b) corresponded to the supramolecular FN–fibrin complexes I–V.

Figure 2.

SDS-agarose immunoblotting of FN and FN–fibrin complexes in plasma of children with RRI. Electrophoresis (300 ng of FN per lane) was undertaken in 1.5% agarose gel under non-reducing conditions and the blots were developed with anti-FN monoclonal antibody (FN30-8; TaKaRa Shuzo Co. Ltd., Japan).¹⁵ (a) Groups: RRI-1 (lane 1), RRI-2 (lane 2), RRI-3 (lane 3); infection (lane 4); normal plasma of child (lane 5) and a commercial plasma FN preparation (lane 6). Molecular masses of standards: Polymers of von Willebrand factor (vWF: 900 kDa, 1350 kDa, 1800 kDa and 2250 kDa) and commercial plasma FN preparation (240 kDa). (b) Reactivity of normal plasma with anti-FN monoclonal antibody (FN) and antiserum directed to human fibrinogen (Fb).^15,17

FN–fibrin complexes I–IV were found in Groups 1–3, the RRI groups, Group 4, the infection group, and Group 5, the normal group, with decreasing frequency of occurrence. In contrast, FN–fibrin complex V occurred exclusively in the plasma of children with RRI, Groups 1–3 (Table 3). The relative amounts of FN–fibrin complexes I–III were significantly higher in the children in Group 1 (RRI-1) (30.95 ± 2.9%, 9.32 ± 5.1%, 3.7 ± 3.6%, respectively) and Group 2 (RRI-2) (30.03 ± 5.9%, 10.54 ± 5.6%, 4.74 ± 4.0%, respectively) than they were in Group 5, the normal group (16.57 ± 9.9%, 1.55 ± 3.7%, 0.64 ± 1.7%, respectively). In addition, the relative amount of complex IV was significantly higher in plasma in the children in Group 2 (RRI-2) (1.69 ± 2.4%) than it was in the plasma of children in the normal group (0.14 ± 0.4%). In contrast, no significant differences were found in the relative amounts of the FN–fibrin complexes between children with RRI in Groups 1–3, and children in Group 4, the infection group, as well as between children with RRI-3 in Group 3, children in Group 4 infection, and children in the normal group, Group 5.

Table 3.

Frequency of occurrence of bands in FN multimers in blood plasma of children suffering from recurrent respiratory infection.

Group/Number of samples		Quantification of FN bands (kDa) in relation to the agarose-immunoblotting pattern shown in Figure 2(a) Frequency of occurrence Relative amount (%) of band
		FN dimer	FN–fibrin complexes
		490 ± 34 ∼500	I 750 ± 20 ∼750	II 1024 ± 39 ∼1000	III 1310 ± 50 ∼1300	IV 1592 ± 38 ∼1600	V 1874 ± 115 ∼1900
1.	RRI-1 n = 20	1 54.01 ± 11.5 P < 0.000	1 30.95 ± 2.9 P < 0.000	1 9.32 ± 5.1 P < 0.000	0.9 3.7 ± 3.6 (18) P < 0.004	0.55 1.28 ± 1.9 (11)	0.25 0.34 ± 0.9 (5)
2.	RRI-2 n = 23	1 52.37 ± 15.0 P < 0.000	1 30.03 ± 5.9 P < 0.000	0.95 10.54 ± 5.6 (22) P < 0.000	0.87 4.74 ± 4.0 (20) P < 0.000	0.69 1.69 ± 2.4 (16) P < 0.02	0.30 0.47 ± 1.1 (7)
3.	RRI-3 n = 21	1 62.67 ± 20.3	1 25.27 ± 9.4	0.71 6.5 ± 6.5 (15)	0.61 2.93 ± 4.3 (13)	0.23 1.27 ± 2.7 (5)	0.19 0.58 ± 1.5 (4)
4.	Infection n = 13	1 59.91 ± 8.2	1 27.86 ± 3.7	1 6.15 ± 3.0	0.69 3.17 ± 1.7 (9)	0.23 0.87 ± 0.2 (3)	Not detected
5.	Normal n = 17	1 80.66 ± 14.3	1 16.57 ± 9.9	0.41 1.55 ± 3.7 (7)	0.17 0.64 ± 1.7 (3)	0.11 0.14 ± 0.4 (2)	Not detected

Note: FN multimers were revealed by SDS-agarose immunoblotting (see Figure 2a). RRI groups were stratified as described in legend to Table 1. The frequency of occurrence is the ratio of the number of samples containing the FN form to the total number of samples. The relative amount of the FN–fibrin band is the percentage of the total number of pixels found in the electrophoresis path and expressed as mean value ± SD. The numbers in parentheses indicate the number of samples which revealed the reduced FN form. P indicates significantly different from the group of normal children and level of significance as calculated by the Kruskal–Wallis and post hoc tests.

Discussion

In the present study, we found that the soluble-supra-molecular plasma FN–fibrin complexes, normally absent in most young healthy individuals, occurred with high frequency in the plasma of children with acute infection and recurrent respiratory infections (RRI). However, their occurrence in the plasma of children with RRI did not correlate with either immunoglobulin concentrations or the number of NK cells.

The SDS-agarose FN immunoblotting pattern (Figure 2(a), Table 3) showed that FN present in the plasma of children with respiratory infection consists of a mixture of native dimeric FN (band of ∼500 kDa, a normal constituent of blood plasma) and a series of supramolecular FN–fibrin complexes visible as the ladder of bands with increasing molecular masses of ∼750-, ∼1000-, ∼1300-, ∼1600 and ∼1900-kDa, which were recognized by anti-FN and anti-fibrinogen antibodies (Figure 2b). As discussed earlier,¹⁵ these FN–fibrin complexes differed from the adjacent one by ∼280 kDa, which corresponded to the mass of one FN reduced subunit crosslinked with a fibrin fragment (Figure 1; Table 2).

The absence of any significant differences in FN–fibrin patterns among children in Groups 1 and 2, and those in Group 4 (Figure 2; Table 3) suggests that the qualitative and quantitative changes in the FN–fibrin complex profile is not related to the ability of the immunological system of children to respond to infection agent stimulation. Moreover, the finding that the plasma FN concentration in the childrens’ groups were in the reference interval (Table 2) suggests that the presence of abnormal FN forms was not related to either the rate of synthesis of FN or the release of the abnormal forms from immunological cells. We believe that the observed pattern may reflect a molecular connection between inflammation and activation of the coagulation cascade. Infection is known to provoke inflammation, and therefore might lead to injury and to malfunctions of inflamed tissue, particularly in children.^18,19 Bacterial products and/or pro-inflammatory cytokines are able to elicit pro-coagulant reactions activated primarily by the tissue factor pathway. Tissue factor binds and activates clotting factor VII, which via factor X results in the generation of thrombin.²⁰ Thus, after activation of a coagulation cascade, during enhanced thrombotic processes, fibrinogen undergoes conversion to fibrin and its fragments, which might form complexes with plasma FN.^6,21 The FN can be covalently crosslinked by factor XIII_a through an isopeptide bond to the carboxyl terminal compact and flexible αC-domain of fibrin,^21,22 creating soluble FN–fibrin hetero-complexes easily revealed by agarose immunoblotting.¹⁵ In contrast, in healthy individuals, FN and fibrinogen circulate in the blood independently (binding sites on fibrinogen for FN are concealed); thus they are unable to interact and therefore the FN–fibrin complexes should not be observed.²¹ Indeed, such FN–fibrin complexes were absent in most of the normal adult samples,¹⁵ although in this study, they were present in a few samples taken from children in the normal group, Group5. However, it should be realized that samples within our normal group were collected from children visiting a doctor for non-infection events. We have previously shown that the FN–fibrin complexes, particularly those with lower molecular masses of ∼750 kDa and ∼1000 kDa, can occur in some normal samples taken from physically healthy individuals and having normal biochemical and haematological parameters.¹⁵

Whilst RRI can cause either acute, chronic or transitional destruction states of the structure of the respiratory epithelium, our results show the molecular consequences of respiratory infections at the level of activation of inflammatory and coagulation systems. The occurrence of a 280-kDa FN–fibrin reduced form and multiple FN–fibrin complexes seems to be a dynamic and transient process related to pathophysiological events probably connected with normal haemostasis.¹⁵ The occurrence of FN–fibrin complexes being evidently associated with inflammatory disease probably reflects FN involvement in the tissue repair process. However, on the other hand, the occurrence of supra-molecular multimers of FN–fibrin complexes carries a risk of disseminated intravascular coagulation in conditions of pro-coagulant states, inhibition of fibrinolysis and consumption of coagulation inhibitors.²³ Such a state might lead to FN–fibrin deposition in the microvasculature and emerging thrombotic micro-angiopathic events. We believe therefore that the findings of this study may have beneficial clinical consequences and prove helpful in the management of therapy, particularly in children with RRI.

Footnotes

Acknowledgements

We thank Dr Leszek Noga for statistical consultations.

Declaration of conflicting interests

None.

Funding

This study was funded by grants from the Wrocław Medical University (PBm6).

Ethical approval

The study was approved by the Commission of Bioethics at Wrocław Medical University (no. KB-445/2012) and conformed to the Helsinki Declaration.

Guarantor

K-PI.

Contributorship

PB was involved in patient recruitment, diagnosis, and written consent. L-PA and K-GD were involved in laboratory work, measuring the parameters, analysis and interpretation of its results. K-PI and L-PA wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript.

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Occurrence of soluble supra-molecular FN–fibrin complexes in the plasma of children with recurrent respiratory infection

Abstract

Objectives

Design and methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Sampling

FN concentration

Western immunoblotting

SDS-agarose FN immunoblotting

Statistics

Results

FN reduced bands

FN multimers

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

Ethical approval

Guarantor

Contributorship

References