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. 2018 Jul 17;17(2):103–111. doi: 10.2450/2018.0043-18

Intravenous immunoglobulin replacement treatment reduces in vivo elastase secretion in patients with common variable immune disorders

Alessandro Prezzo 1, Filomena M Cavaliere 1, Cinzia Milito 1, Caterina Bilotta 1, Metello Iacobini 2, Isabella Quinti 1,✉
PMCID: PMC6476744  PMID: 30036181

Abstract

Background

Intravenous immunoglobulin (IVIg) treatment partially replaces antibody defects and modulates innate and adaptive immune cells in patients with primary antibody deficiencies.

Materials and methods

This study was focused on the evaluation of the effects of in vivo IVIg administration on neutrophils from patients with common variable immune disorders (CVID). We examined polymorphonuclear neutrophil (PMN) phagocytosis, PMN oxidative burst, release of neutrophil elastase, serum level of interleukin-8 and PMN expression of CXCR1, CD11c and CD66b.

Results

CVID patients on chronic IVIg treatment had reduced elastase release, but normal expression of CXCR1, CD66b and CD11c receptors on PMN, normal phagocytic ability and normal secretion of interleukin-8. We found that IVIg infusions rapidly reduced the serum level of interleukin-8, the expression of its receptor, CXCR1, and the release of neutrophil elastase, suggesting that IVIg exert a dampening effect on neutrophil activity. In contrast, IVIg infusions did not alter neutrophil phagocytosis or the expression of the other receptors analysed.

Discussion

These findings add further information regarding the anti-inflammatory role of immunoglobulins and suggest additional benefits in keeping with recent attempts to use new therapies targeting neutrophil inflammation.

Keywords: common variable immune disorders, intravenous immunoglobulins, polymorphonuclear neutrophils, neutrophil elastase, interleukin-8

Introduction

Severe and recurrent infections are common at the time of diagnosis and during the course of diseases in patients with common variable immune disorders (CVID)1–3. CVID patients are characterised by failure to raise an efficient antibody response and by dysregulation of several other elements of the immune system, including naïve CD4 T cells, antigen-specific T cells, cytokine secretion, monocytes, natural killer cells and dendritic cells1,4,5.

Airway infections, such as sinusitis and pneumonia, are common conditions in patients with CVID and lead to lung infection-related structural changes such as bronchiectasis6–8. The production of reactive oxygen species (ROS) and proteases plays an important role in the pathogenesis of inflammatory diseases and their excessive release might contribute to tissue damage in patients with chronic lung diseases8–10. Intravenous or subcutaneous administration of immunoglobulins is a life-saving treatment for CVID patients, enabling partial control of the infectious episodes11–15. The antibody replacement function of intravenous immunoglobulin (IVIg) infusions in protecting against infections appears intuitive, but their immunomodulatory and anti-inflammatory activities are less obvious and probably depend on several mechanisms16–19. Immunomodulatory and anti-inflammatory activities are important not only in the treatment of autoimmune diseases but also in patients suffering from immunodeficiencies20.

In this study, in order to estimate the strength of the immune response mediated by polymorphonuclear neutrophils (PMN) in CVID, we analysed PMN phagocytosis, oxidative burst and release of neutrophil elastase (NE). Moreover, we focused our attention on the IL-8/CXCR1 axis through evaluation of the serum level of interleukin-8 (IL-8/CXCL8), a pro-inflammatory chemokine involved in neutrophil recruitment and degranulation, and analysis of expression of the IL-8 receptor (CXCR1)21,22. In addition, we evaluated the expression of CD11c and CD66b on neutrophils. CD11c is an integrin molecule member of αXβ2 receptor involved in adherence and phagocytosis of complement-coated particles23,24. CD66b is a marker of granulocyte activation, involved in neutrophil cell adhesion, migration, pathogen binding and secretion of preformed IL-825–27. Thus, the simultaneous analysis of these receptors may provide clues on the activation status of PMN from CVID patients.

We have previously shown that in vivo IVIg treatment exerts an anti-inflammatory effect in CVID and X-linked agammaglobulinemia by reducing monocyte activities28,29. IVIg infusions do not affect PMN migration, ROS production or the expression of several markers of PMN activation29,30. However, IVIg do induce a dose-dependent degranulation of neutrophils when added in vitro to whole blood or purified PMN from healthy donors31. Since excessive NE release and ROS production are involved in the pathogenesis of tissue damage32–35, we verified the effects of in vivo IVIg administration on NE release and ROS production in CVID patients receiving chronic replacement therapy.

Materials and methods

Patients and controls

CVID were diagnosed according to the criteria established by the European Society for Immune Deficiencies (http://www.esid.org). Twenty-two adult CVID patients (mean age: 52.1±15.9 years; age range, 27–80 years) and 15 healthy donors (mean age: 37.4±8.9 years; age range, 26–59 years) were enrolled in the study. Patients were in a clinically stable condition without fever and not hospitalised at the time of the study. None of the patients was on steroids or immunosuppressive drugs at the time of the study.

All CVID patients were on replacement treatment, which had been given for an average period of 16.5 years (range, 5–30 years). The median IVIg dose (5% solution) was 285 mg/kg (range, 190–400 mg/kg) in a single session with a cumulative monthly dose of 400–600 mg/kg (Table I). The infusion time ranged from 2 to 3 hours, with the infusion speed established according to individual tolerability.

Table I.

Demographic and clinical data of the patients with common variable immune disorders.

Patient Age/sex Serum IgG* (mg/dL) Neutrophils (×109/L) IVIg dose (mg/kg) administered in a single session Comorbidities
1 65/F 553 3.42 310 Conjunctivitis, dermatitis
2 47/F 822 2.73 350 Respiratory tract infections
3 32/M 756 4.77 310 Splenomegaly, autoimmune thrombocytopenia
4 64/M 608 3.12 280 Splenomegaly, chronic gastritis
5 59/M 680 1.80 230 Respiratory tract infections, splenomegaly
6 41/F 445 1.87 190 Pancytopenia, respiratory tract infections
7 25/M 812 4.08 240 Ulcerative recto-colitis
8 34/M 860 4.87 340 Respiratory tract infections, COPD
9 50/M 770 2.98 310 Respiratory tract infections
10 68/M 635 5.02 320 Vitiligo, respiratory tract infections, COPD
11 36/M 448 2.93 200 Splenomegaly
12 72/F 638 4.55 360 Respiratory tract infections
13 80/M 810 3.27 220 COPD, conjunctivitis
14 42/F 698 1.98 230 Liver disease, respiratory tract infections
15 68/F 584 2.03 380 Celiac disease, chronic gastritis
16 43/M 680 6.92 400 Respiratory tract infections, COPD
17 27/F 545 3.44 360 COPD, chronic gastritis
18 48/M 624 5.65 220 Respiratory tract infections
19 54/F 882 2.14 230 Respiratory tract infections, COPD
20 64/F 450 1.85 240 Respiratory tract infections
21 56/M 636 3.26 360 Respiratory tract infections, chronic gastritis
22 72/F 740 3.02 190 Splenomegaly, dermatitis
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*

Serum IgG values refer to pre-infusion levels.

F: female; M: male; COPD: chronic obstructive pulmonary disease.

This study was approved by the Ethics Committee of the Sapienza University of Rome. All participants gave written informed consent prior to inclusion.

Preparation of blood samples

Heparinised whole blood samples were collected from 15 healthy donors and 22 patients with CVID immediately before administration of IVIg and 1 hour after the end of the infusion. These time points were chosen based on our previous observations28, taking into consideration that the greatest increase of several cytokines occurred within 1 hour after IVIg infusion36. Total peripheral blood monocyte and neutrophil counts were determined from full blood cell counts and the white blood cell differential. In order to evaluate circulating cells without the harm of cell loss related to the density gradient centrifugation procedure, peripheral blood red cells were lysed using lysing buffer (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA). Samples were washed twice before being stained with various combinations of fluorochrome-labelled antibodies. All antibodies were obtained from Becton Dickinson Biosciences. Flow cytometric analysis was done with a FACSCalibur instrument (Becton Dickinson Biosciences) using CellQuest (Becton Dickinson Biosciences) and FlowJo (TreeStar, Ashland, OR, USA) software. The stability and sensitivity of the cytometer were checked before each acquisition session by using microbeads designed to control the efficiency, the coefficient of variation of scatter and fluorescence signals and the time delay calibration (Nile Red Fluorescent particles and Calibrate APC Beads, all from Becton Dickinson Biosciences).

Results are expressed as geometric mean fluorescence intensity (MFI) of any given marker within the defined population, with 30,000 events counted per sample.

Polymorphonuclear neutrophil elastase assay

PMN elastase was quantified by Human PMN Elastase Platinum enzyme-linked immunosorbent assay (Affymetrix eBioscience, Cambridge, England) on plasma collected from unstimulated and stimulated blood samples by centrifugation for 15 min at 1,000 × g, diluted and tested in duplicate. One hundred microlitres of each plasma sample were added to a microwell plate coated with polyclonal antibody to human PMN elastase and incubated at room temperature for 60 min. After washing, samples were incubated with TMB Substrate Solution at room temperature for 20 min in the dark before the addition of 50 μL of stop solution to arrest the reaction. The absorbance was determined at 450 nm on a microplate reader Multiskan™ FC Microplate Photometer (ThermoScientific, Waltham, MA, USA). Concentrations are expressed as ng/mL.

Analysis of receptor expression on polymorphonuclear neutrophils

Whole blood samples were first treated to lyse red blood cells and then washed twice and stained at 4 °C for 30 min with combinations of fluorochrome-labelled antibodies. Samples were washed, suspended in ice-cold phosphate-buffered saline and analysed by a four-color flow cytometry single platform. The surface expression of CXCR1, CD66b and CD11c was evaluated on PMN samples and analysed by flow cytometry. PMN were identified by forward and side scatter characteristics after gating on CD15-positive events.

Polymorphonuclear neutrophil stimulation by Escherichia coli

One hundred microlitres of whole blood collected before and after IVIg administration were added to 20 μL of pre-cooled opsonised, unlabelled whole Escherichia coli (E. coli) at a concentration of 1–2×109/mL (Glycotope Biotechnology, Heidelberg, Germany). Samples were incubated in a water bath for 20 min at 37 °C and erythrocytes were lysed for 15 min at room temperature. Stimulated cells were stained at 4 °C for 30 min with various combinations of fluorochrome-labelled antibodies to CXCR1, CD66b and CD11c in order to evaluate expression of these receptors.

Cytokine plasma dosage

IL-8 was measured using a commercially available enzyme-linked immunosorbent assay kit (4A Biotech Co, Beijing, China). Blood samples were stimulated with N-formylmethionyl-leucyl-phenylalanine (fMLP), E. coli and opsonised E. coli. Plasma was collected from unstimulated and stimulated blood samples by centrifugation for 15 min at 1,000 × g, diluted and tested in duplicate.

One hundred microlitres of each plasma sample were added to a microwell plate coated with monoclonal antibody to human IL-8 and incubated at 37 °C for 90 min. After washing, samples were incubated with biotin-conjugate solution at 37 °C for 60 min. Each well was then washed several times and incubated with 100 μL of streptavidin-horse radish peroxidase at 37 °C for 30 min. After washing, 100 μL of substrate solution were added to each well and incubated for 10–20 min a 37 °C. The reaction was then halted by the addition of 100 μL of stop solution. The absorbance was determined at 450 nm on a microplate reader Multiskan™ FC Microplate Photometer (ThermoScientific). Concentrations are expressed as pg/mL.

Polymorphonuclear neutrophil phagocytosis

PMN phagocytosis was determined using a PHAGOTEST assay (Glycotope Biotechnology). Whole blood samples (100 μL) were incubated in a water bath for 10 min at 37 °C with opsonised E. coli (2×109/mL) labelled with fluorescein isothiocyanate (FITC). As a negative control, 100 μL of each sample were incubated in an ice bath for 10 min with opsonised FITC-labelled E. coli (2×109/mL). As another control, the same experiment was done using non-opsonised FITC-labelled E. coli (2×109/mL). We analysed phagocytosis at different time points (5, 10, 15, 20 min) after incubation with FITC-labelled E. coli, determining the percentage and MFI of granulocytes having performed phagocytosis by flow cytometry using a blue-green excitation light (488 nm argon-ion laser).

Polymorphonuclear neutrophil oxidative burst

The leucocyte oxidative burst was analysed using the PHAGOBURST assay (Glycotope Biotechnology). Whole blood samples (100 μL) were incubated in a water bath for 20 min at 37 °C with opsonised E. coli (1–2×109/mL) or a wash solution as a negative control. The percentage and MFI of cells that produced ROS were determined. The intracellular production of superoxide anions and hydrogen peroxide in PMN in response to phagocytosis of bacteria was tested by using the fluorescence probe dihydrorhodamine 123 (DHR 123). We repeated the analysis of the oxidative burst using non-opsonised E. coli (1.5×109/mL) as a control, in order to exclude Fcg receptor (FcgR)-independent phagocytosis. Oxidation leads to fluorescence which can be detected by flow cytometry using a blue-green excitation light (488 nm argon-ion laser).

Statistical analysis

Data were analysed using the Mann-Whitney U test for two unpaired groups or Wilcoxon’s test for paired samples. Statistical computations were performed with StatView 5.0.1 software (SAS Institute, Cary, NC; USA) and p values equal to or less than 0.05 are considered statistically significant.

Results

Polymorphonuclear neutrophil elastase release and the in vivo effect of intravenous immunoglobulin infusion

Activated neutrophils secrete the contents of their granules into the extracellular space during chronic neutrophilic inflammation. Primary azurophilic granules contain a number of neutrophil serine proteases, including NE37. Since the amount of NE protease released reflects the strength of the neutrophil response to pathogens, we evaluated the serum levels of NE in CVID patients before and after IVIg infusion. We also evaluated the amount of NE released after ex vivo stimulation of whole blood with fMLP, E. coli and opsonised E. coli, in order to avoid artefacts caused by the isolation procedure and to reproduce an in vivo infection more faithfully.

CVID patients had a lower NE serum level compared to healthy donors (CVID: 301.8±75.7 ng/mL vs healthy donors: 420.4±150.2 ng/mL, p=0.003). After each stimulus, the release of NE increased equally in healthy donors and CVID patients (Figure 1). However, the levels of NE reached in CVID patients after ex vivo stimulation with opsonised E. coli were lower than those observed in healthy donors (CVID: 1,097.1±57.8 ng/mL vs healthy donors: 1,386.6±60.4 ng/mL, p=0.027).

Figure 1.

Figure 1

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Effect of intravenous immunoglobulin administration on in vivo polymorphonuclear neutrophil elastase release in patients with common variable immune disorders.

Neutrophil elastase (NE) was quantified by enzyme-linked immunosorbent assay on plasma that was unstimulated or stimulated with fMLP, E. coli and opsonised E. coli. The NE release in unstimulated plasma was reduced in patients with common variable immune disorders (CVID) compared to healthy donors (HD). NE release was defective in CVID patients after stimulation with opsonised E. coli, while it was normal after fMLP and E. coli stimulation. After IVIg infusion, NE release was further reduced in unstimulated plasma and after stimulation with opsonised E. coli, while it remained unaltered after the other stimuli. Concentrations are expressed in ng/mL.

*p=0.003; **p=0.027; ***p=0.002.

fMLP: N-formylmethionyl-leucyl-phenylalanine; E. coli: Escherichia coli.

IVIg infusion induced a rapid drop of NE levels in patients’ sera (from 301.8±75.7 ng/mL to 161.8±56.7 ng/mL, p=0.003). Furthermore, the NE release after ex vivo stimulation with opsonised E. coli was halved after IVIg infusion compared to that observed before infusion (from 1,097.1±57.8 to 598.3±36.1 ng/mL, p=0.002) while it remained unchanged when other stimuli were used (Figure 1 and On-line supplementary content, Table SI).

Polymorphonuclear neutrophil surface receptors and the in vivo effect of intravenous immunoglobulin infusion

CXCR1 responds to IL-8, strongly influencing the activation of PMN, CD11c is involved in cell adherence and phagocytosis while CD66b is involved in neutrophil adhesion, migration and pathogen binding. As shown in Figure 2 and On-line supplementary content, Table SII, PMN from CVID and healthy donors showed similar levels of CXCR1, CD11c and CD66b expression. As expected29,38, stimulation with opsonised E. coli reduced the expression of CXCR1 and induced overexpression of CD11c and CD66b on PMN from CVID patients and healthy donors (On-line supplementary content, Table SII and Figure 2A–C).

Figure 2.

Figure 2

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CXCR1, CD66b and CD11c expression on polymorphonuclear neutrophils from patients with common variable immune disorders before and after intravenous immunoglobulin infusion.

Whole blood samples unstimulated and stimulated with opsonised E. coli were analysed for the expression of (A) CXCR1, (B) CD66b and (C) CD11c before and after infusion of intravenous immunoglobulins IVIg infusion. The expression of all surface receptors was analyzed by flow cytometry. E. coli stimulation induced overexpression of CD66b and CD11c, while it decreased the expression of CXCR1. After IVIg infusion, the expression of (B) CD66b and (C) CD11c receptors remained unchanged, while (A) CXCR1 expression decreased (*p=0.001). Results are expressed as mean fluorescence intensity. Histograms denote mean values and bars standard deviation. Statistical significance, determined by the non-parametric Mann-Whitney and Wilcoxon’s signed rank tests, is indicated as p values. HD: healthy donors; CVID: common variable immune disorders; IVIg: intravenous immunoglobulins; MFI: mean fluorescence intensity; E. coli: Escherichia coli.

One hour after the IVIg infusion, CXCR1 expression on unstimulated PMN was reduced, while CD66b and CD11c expression remained unaltered (Figure 2A–C). The IVIg infusion did not alter the expression of PMN receptors in response to opsonised E. coli (Online Supplementary Table II and Figure 2A–C).

Interleukin-8 levels and the in vivo effect of intravenous immunoglobulin infusion

IL-8 activates neutrophils via the chemokine receptors CXCR1 and CXCR2, promoting the killing ability of neutrophils22. As shown in Figure 3, IL-8 serum levels from unstimulated and stimulated whole blood were similar in CVID patients and healthy donors (CVID: 19.6±4.6 pg/mL vs healthy donors: 17±6.8 pg/mL, p=ns). Stimulation with opsonised E. coli induced similar increases of IL-8 secretion in CVID patients and healthy donors (CVID: from 19.6±4.6 pg/mL to 112. ±71.8 pg/mL p=0.02, healthy donors: from 17±6.8 pg/mL to 107±65 pg/mL, p=0.02), while stimulation with fMLP and E. coli did not cause any increase of IL-8 levels in either CVID patients or healthy donors (Figure 3).

Figure 3.

Figure 3

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Plasma levels of interleukin-8 in patients with common variable immune disorders.

IL-8 production was measured by an enzyme-linked immunosorbent assay kit in unstimulated plasma and plasma stimulated with fMLP, E. coli and opsonised E. coli. In unstimulated plasma IL-8 levels were similar in CVID patients and HD. Following stimulation with opsonised E. coli, we found the same increased levels of IL-8 in CVID patients and HD. After IVIg infusion, IL-8 levels decreased in unstimulated plasma. Concentrations are expressed in pg/mL. The histograms represent mean values and the bars standard deviation. Statistical significance, determined by the non-parametric Mann-Whitney and Wilcoxon’s signed rank tests, is indicated as a p value. *p=0.035.

HD: healthy donors; CVID: common variable immune disorders; IVIg: intravenous immunoglobulins; IL-8: interleukin-8; fMLP: N-formylmethionyl-leucyl-phenylalanine; E. coli: Escherichia coli.

One hour after IVIg infusion, we observed a significant reduction of IL-8 levels only on unstimulated CVID PMN (from 19.6±4.6 pg/mL to 15.8±1.6 pg/mL, p=0.01) (Figure 3).

Polymorphonuclear neutrophil phagocytosis, production of reactive oxygen species and the in vivo effect of intravenous immunoglobulin infusion

Given that soon after IVIg infusion, NE release, IL-8 secretion and the expression of CXCR1 were reduced, it became crucial to verify the integrity of the PMN killing ability in CVID patients receiving chronic IVIg treatment.

We evaluated the phagocytic process using FITC-labelled opsonised E. coli and non-opsonised FITC-labelled E. coli as a control with the aim of testing the ability of PMN to internalise bacteria through FcgR. In addition, we tested ROS production using unlabelled opsonised E. coli and unlabelled, non-opsonised E. coli as a control.

PMN from CVID patients and healthy donors, gated on the basis of forward and side scatter characteristics (Figure 4A), showed comparable phagocytosis (CVID: 607±206.9 MFI vs healthy donors: 658.8±178.9 MFI, p=ns) (Figure 4B). One hour after IVIg infusion, phagocytosis remained unaltered (from 607±206.9 MFI to 614.3±169.5 MFI, p=ns) (Figure 4B). Phagocytosis was also evaluated at various time points after activation, and showed a similar trend in CVID patients and healthy donors (Figure 4C).

Figure 4.

Figure 4

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Polymorphonuclear neutrophil phagocytosis and oxidative burst in patients with common variable immune disorders. The analysis of phagocytosis and oxidative burst by flow cytometry was performed (A) gating on PMN using forward scatter (FSC) and side scatter (SSC) characteristics. Leucocyte phagocytosis was quantified by incubating whole blood samples with FITC-labelled E. coli: the percentage of cells having performed phagocytosis was determined from the number of ingested bacteria and results are expressed as MFI. (B) CVID patients and HD showed comparable phagocytosis and this remained unchanged after IVIg administration. (C) The phagocytosis observed at various times points (5, 10, 15 and 20 min) was similar in CVID patients and HD. (D) The oxidative burst of leucocytes was evaluated by incubating whole blood samples with opsonised E. coli: CVID patients and HD showed a comparable PMN oxidative burst. The oxidative burst remained unchanged after IVIg administration. (E) The oxidative burst reached a maximum intensity in CVID patients after 20 min, while HD showed the maximum intensity of oxidative burst 15 min after E. coli incubation (p=0.006). Results are expressed as MFI. Histograms represent mean values and bars standard deviation. Statistical significance, as determined by the non-parametric Wilcoxon’s signed rank test, is indicated as a p value.

PMN: polymorphonuclear neutrophils; HD: healthy donors; CVID: common variable immune disorders; IVIg: intravenous immunoglobulins; MFI: mean fluorescence intensity; E. coli: Escherichia coli.

The oxidative burst induced by E. coli was similar in CVID patients and healthy donors (CVID: 519.8±203.1 MFI vs healthy donors 508.8±137.7 MFI, p=ns) (Figure 4D). ROS production 1 hour after IVIg infusion was comparable to that observed prior to the infusion (from 519.8±203.1 MFI to 539.5±301.4 MFI, p=ns) (Figure 4D).

We also analysed the oxidative burst induced by E. coli at different time points (5, 10, 15 and 20 min). Compared to cells from CVID patients, those from healthy donors showed higher ROS production 15 min after incubation with E. coli (CVID: 390.2±70.7 MFI vs healthy donors 475.6±52.4 MFI, p=0.006) (Figure 4E). At every time point analysed 1 hour after the IVIg infusion, the ROS production was comparable to that observed prior to the infusion (Figure 4E).

Discussion

The administration of low, replacement doses of immunoglobulins is the standard therapy for CVID patients13,14. IVIg exert both immunomodulatory and anti-inflammatory effects and reduce the frequency of acute respiratory infections16–20. However, individuals with CVID often have bronchiectasis, which is believed to be a neutrophil driven disorder39,40. Neutrophils are the most abundant cells in the airway fluid of patients with bronchiectasis. During inflammation, neutrophils rapidly migrate to the site of inflammation where they release granules containing proteolytic enzymes, antimicrobial peptides and ROS39,40, playing a protective role during infection. Excessive degranulation, however, induces tissue damage41,42. Indeed neutrophil granules contain the pro-inflammatory protease elastase and it has been shown that the amount of elastase correlates with the severity of lung dysfunction, contributing to the progression of bronchiectasis37,43,44. In the light of this evidence, elastase is a leading candidate for therapeutic manipulation in patients with bronchiectasis45. The previous demonstration that IVIg induced dose-dependent degranulation in vitro suggested that this treatment could have a negative impact on the pathogenesis of lung tissue damage31. Thus, we verified in CVID patients whether in vivo IVIg administration increases NE and ROS production.

We found that CVID patients who had been receiving IVIg infusions for several years had lower plasma levels of NE than healthy donors, even after E. coli stimulation. This observation suggests that PMN from CVID patients are less prone to degranulation than those from healthy donors. However, the phagocytic ability and expression of CXCR1, CD66b and CD11c receptors on PMN appeared normal. Consistently, the prompt overexpression of CD11c and CD66b and the reduction of CXCR1 expression observed after stimulation with opsonised E. coli seem to confirm that PMN normally express those receptors involved in their activation29,30,38. We also observed normal IL-8 levels in CVID patients, consistent with the normal PMN function found in this series of experiments and in our previous studies29,30.

In contrast with previous in vitro evidence31, in this study we show that in vivo IVIg infusion induces a rapid and profound reduction of NE degranulation. This effect could contribute to the anti-inflammatory properties of IVIg and to an improvement of pulmonary inflammatory conditions. This finding opens a new scenario for therapeutic strategies aimed at reducing excessive NE release. We also observed a slight decrease of the plasma level of IL-8 and reduced expression of its receptor, CXCR1, on PMN after IVIg infusions, although we found normal phagocytic activity. It can, therefore, be speculated that a weakening of the IL-8/CXCR1 axis might contribute to the reduction of NE release without affecting FcgRs-mediated activities (Figure 5). Natural autoantibodies, such as anti-Siglec 9 antibodies, contained in IVIg preparations might contributed to the anti-inflammatory effects of IVIg36,46–48. Moreover, since it has been shown that sialic acid dampened the activity of neutrophils through specific binding to Siglec 948–53, it is possible that the sialylated portion of IVIg contributes to the decrease of elastase degranulation observed after IVIg infusions (Figure 5). More recently, there have been reports of other IVIg-, but not Fcg-, mediated mechanisms acting on neutrophils in vitro, such as those mediated by pepsin54.

Figure 5.

Figure 5

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Representation of the effect of intravenous immunoglobulins on elastase release.

Our model hypothesises that IVIg act in CVID patients by dampening the IL-8/CXCR1 axis, thus reducing elastase release. In particular, the autoantibodies present in IVIg preparations might reduce IL-8 levels and the simultaneous reduction of CXCR1 could lead to reduced cell activation. The activation of Siglec 9 receptors, by the sialylated portion of immunoglobulins or by anti-Siglec 9 autoantibodies, is also possible. Siglec 9 activation would transduce an inhibitory signal inside the cell, limiting neutrophil activation. Each of these mechanisms probably contributes to the reduction of neutrophils degranulation.

IVIg: intravenous immunoglobulins; IL-8: interleukin-8.

Conclusions

This study has revealed a possible novel anti-inflammatory role of replacement IVIg therapy. Although the main functions of PMN from CVID patients are well preserved, we found impaired elastase degranulation. In vivo IVIg administration at low dosages induced a further rapid decrease of NE release associated with a reduction of IL-8/CXCR1 levels. These results suggest that the impairment of the IL-8/CXCR1 axis following IVIg infusions may play a role in reducing NE release in CVID patients. Although further and more detailed studies are needed, we suggest that this is an additional protective role of IVIg in neutrophil-driven inflammation.

On-line Supplementary Content

Footnotes

Authorship contributions

AP and FMC contributed equally to this study.

AP, FMC and CB designed the study and conducted the laboratory tests included in the study. AP and FMC collected and analysed data and prepared the manuscript. IQ and MI edited and reviewed the manuscript with contributions from CM and Albert Farrugia.

All Authors critically reviewed and revised the manuscript drafts, approved the final version of the manuscript and take responsibility for the integrity of the data and accuracy of data analysis.

The Authors declare no conflicts of interest.

Funding

This work was supported in part by the University of Rome and by the Jeffrey Modell Foundation.

References

  • 1.Chapel H, Cunningham-Rundles C. Update in understanding common variable immunodeficiency disorders (CVIDs) and the management of patients with these conditions. Br J Haematol. 2009;6:709–27. doi: 10.1111/j.1365-2141.2009.07669.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Quinti I, Di Pietro C, Martini H, et al. Health related quality of life in common variable immunodeficiency. Yonsei Med J. 2012;53:603–10. doi: 10.3349/ymj.2012.53.3.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bonilla FA, Barlan I, Chapel H, et al. International consensus document (ICON): common variable immunodeficiency disorders. J Allergy Clin Immunol Pract. 2016;4:38–59. doi: 10.1016/j.jaip.2015.07.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Saikia B, Gupta S. Common variable immunodeficiency. Indian J Pediatr. 2016;83:338–44. doi: 10.1007/s12098-016-2038-x. [DOI] [PubMed] [Google Scholar]
  • 5.Cunningham-Rundles C, Bodian C. Common variable immunodeficiency: clinical and immunological features of 248 patients. Clin Immunol. 92:34–48. doi: 10.1006/clim.1999.4725. 199. [DOI] [PubMed] [Google Scholar]
  • 6.Reisi M, Azizi G, Kiaee F, et al. Evaluation of pulmonary complications in patients with primary immunodeficiency disorders. Eur Ann Allergy Clin Immunol. 2017;49:122–8. [PubMed] [Google Scholar]
  • 7.Mooney D, Edgar D, Einarsson G, et al. Chronic lung disease in common variable immune deficiency (CVID): a pathophysiological role for microbial and non-B cell immune factors. Crit Rev Microbiol. 2017;43:508–19. doi: 10.1080/1040841X.2016.1268568. [DOI] [PubMed] [Google Scholar]
  • 8.Mizgerd JP. Acute lower respiratory tract infection. N Engl J Med. 2008;358:716–27. doi: 10.1056/NEJMra074111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.de Gracia J, Vendrell M, Alvarez A, et al. Immunoglobulin therapy to control lung damage in patients with common variable immunodeficiency. Int Immunopharmacol. 2004;4:745–53. doi: 10.1016/j.intimp.2004.02.011. [DOI] [PubMed] [Google Scholar]
  • 10.Conner EM, Grisham MB. Inflammation, free radicals, and antioxidants. Nutrition. 1996;12:274–7. doi: 10.1016/s0899-9007(96)00000-8. [DOI] [PubMed] [Google Scholar]
  • 11.De Ranieri D, Fenny NS. Intravenous immunoglobulin in the treatment of primary immunodeficiency diseases. Pediatr Ann. 2017;46:e8–e12. doi: 10.3928/19382359-20161213-03. [DOI] [PubMed] [Google Scholar]
  • 12.Kerr J, Quinti I, Eibl M, et al. Is dosing of therapeutic immunoglobulins optimal? A review of a three-decade long debate in Europe. Front Immunol. 2014;5:1–19. doi: 10.3389/fimmu.2014.00629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Abolhassani H, Sagvand BT, Shokuhfar T, et al. A review on guidelines for management and treatment of common variable immunodeficiency. Exp Rev Clin Immunol. 2013;9:561–74. doi: 10.1586/eci.13.30. [DOI] [PubMed] [Google Scholar]
  • 14.Quinti I, Soresina A, Agostini C, et al. Prospective study on CVID patients with adverse reactions to intravenous or subcutaneous IgG administration. J Clin Immunol. 2008;28:263–7. doi: 10.1007/s10875-007-9169-9. [DOI] [PubMed] [Google Scholar]
  • 15.Kivity S, Katz U, Daniel N, et al. Evidence for the use of intravenous immunoglobulins - a review of the literature. Clin Rev Allergy Immunol. 2010;38:201–69. doi: 10.1007/s12016-009-8155-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Paquin-Proulx D, Sandberg JK. Persistent immune activation in CVID and the role of IVIg in its suppression. Front Immunol. 2014;5:637. doi: 10.3389/fimmu.2014.00637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kazatchkine MD, Kaveri SV. Immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin. N Engl J Med. 2001;345:747–55. doi: 10.1056/NEJMra993360. [DOI] [PubMed] [Google Scholar]
  • 18.Nimmerjahn F, Ravetch JV. Anti-inflammatory actions of intravenous immunoglobulin. Annu Rev Immunol. 2008;26:513–33. doi: 10.1146/annurev.immunol.26.021607.090232. [DOI] [PubMed] [Google Scholar]
  • 19.Bichuetti-Silva DC, Furlan FP, Nobre FA, et al. Immediate infusion-related adverse reactions to intravenous immunoglobulin in a prospective cohort of 1765 infusions. Int Immunopharmacol. 2014;23:442–6. doi: 10.1016/j.intimp.2014.09.015. [DOI] [PubMed] [Google Scholar]
  • 20.Matucci A, Maggi E, Vultaggio A. Mechanisms of action of Ig preparations: immunomodulatory and anti-inflammatory effects. Front Immunol. 2015;5:690. doi: 10.3389/fimmu.2014.00690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.David JM, Dominguez C, Hamilton DH, et al. The IL-8/IL-8R axis: a double agent in tumor immune resistance. Vaccines (Basel) 2016;4:22. doi: 10.3390/vaccines4030022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ha H, Debnath B, Neamati N. Role of the CXCL8-CXCR1/2 axis in cancer and inflammatory diseases. Theranostics. 2017;7:1543–88. doi: 10.7150/thno.15625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Smith SL, Eyre S, Yarwood A, et al. Investigating CD11c expression as a potential genomic biomarker of response to TNF inhibitor biologics in whole blood rheumatoid arthritis samples. Arthritis Res Ther. 2015;17:359. doi: 10.1186/s13075-015-0868-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Krystufkova O, Mann H, Hulejova H, et al. Enhanced expression of CD11c on non-classical CD16+ peripheral blood monocytes in early rheumatoid arthritis. Ann Rheum. 2014;73:A20–A21. [Google Scholar]
  • 25.Kuijpers TW, van der Schoot CE, Hoogerwerf M, et al. Cross-linking of the carcinoembryonic antigen-like glycoproteins CD66 and CD67 induces neutrophil aggregation. J Immunol. 1993;151:4934–40. [PubMed] [Google Scholar]
  • 26.Nair KS, Zingde SM. Adhesion of neutrophils to fibronectin: role of the cd66 antigens. Cell Immunol. 2001;208:96–106. doi: 10.1006/cimm.2001.1772. [DOI] [PubMed] [Google Scholar]
  • 27.Schröder AK, Uciechowski P, Fleischer D, et al. Crosslinking of CD66B on peripheral blood neutrophils mediates the release of interleukin-8 from intracellular storage. Hum Immunol. 2006;67:676–82. doi: 10.1016/j.humimm.2006.05.004. [DOI] [PubMed] [Google Scholar]
  • 28.Cavaliere FM, Prezzo A, Conti V, et al. Intravenous immunoglobulin replacement induces an in vivo reduction of inflammatory monocytes and retains the monocyte ability to respond to bacterial stimulation in patients with common variable immunodeficiencies. Int Immunopharmacol. 2015;28:596–603. doi: 10.1016/j.intimp.2015.07.017. [DOI] [PubMed] [Google Scholar]
  • 29.Cavaliere FM, Prezzo A, Bilotta C, et al. The lack of BTK does not impair monocytes and polymorphonuclear cells functions in X-linked agammaglobulinemia under treatment with intravenous immunoglobulin replacement. PLoS One. 2017;19(12):e0175961. doi: 10.1371/journal.pone.0175961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Prezzo A, Cavaliere FM, Bilotta C, et al. Intravenous immunoglobulin replacement treatment does not alter polymorphonuclear leukocytes function and surface receptors expression in patients with common variable immunodeficiency. Cell Immunol. 2016;306–307:25–34. doi: 10.1016/j.cellimm.2016.05.006. [DOI] [PubMed] [Google Scholar]
  • 31.Teeling JL, De Groot ER, Eerenberg AJ, et al. Human intravenous immunoglobulin (IVIG) preparations degranulate human neutrophils in vitro. Clin Exp Immunol. 1998;114:264–70. doi: 10.1046/j.1365-2249.1998.00697.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Mittal M, Siddiqui MR, Tran K, et al. Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal. 2014;20:1126–67. doi: 10.1089/ars.2012.5149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ciencewicki J, Trivedi S, Kleeberger SR. Oxidants and the pathogenesis of lung diseases. J Allergy Clin Immunol. 2008;122:456–70. doi: 10.1016/j.jaci.2008.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.King PT. Inflammation in chronic obstructive pulmonary disease and its role in cardiovascular disease and lung cancer. Clin Transl Med. 2015;4:26. doi: 10.1186/s40169-015-0068-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Nee WM. Pathogenesis of chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2005;2:258–66. doi: 10.1513/pats.200504-045SR. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Aukrust P, Frøland SS, Liabakk NB, et al. Release of cytokines, soluble cytokine receptors, and interleukin-1 receptor antagonist after intravenous immunoglobulin administration in vivo. Blood. 1994;84:2136–43. [PubMed] [Google Scholar]
  • 37.Mårdh CK, Root J, Uddin M, et al. Targets of neutrophil influx and weaponry: therapeutic opportunities for chronic obstructive airway disease. J Immunol Res. 2017 doi: 10.1155/2017/5273201. 5273201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Tikhonov I, Doroshenko T, Chaly Y, et al. Down-regulation of CXCR1 and CXCR2 expression on human neutrophils upon activation of whole blood by S. aureus is mediated by TNF-α. Clin Exp Immunol. 2001;125:414–22. doi: 10.1046/j.1365-2249.2001.01626.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Daheshia M, Prahl JD, Carmichael JJ, et al. The immune response and Its therapeutic modulation in bronchiectasis. Pulm Med. 2012;2012 doi: 10.1155/2012/280528. 280528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Chalmers JD, Hill AT. Mechanisms of immune dysfunction and bacterial persistence in non-cystic fibrosis bronchiectasis. Mol Immunol. 2013;55:27–34. doi: 10.1016/j.molimm.2012.09.011. [DOI] [PubMed] [Google Scholar]
  • 41.Stockley R, Bayley D, Hill S, et al. Assessment of airway neutrophils by sputum colour: correlation with airways inflammation. Thorax. 2001;56:366–72. doi: 10.1136/thorax.56.5.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Stockley RA, Hill SL, Morriso HM, et al. Elastolytic activity of sputum and its relation to purulence and to lung function in patients with bronchiectasis. Thorax. 1984;39:408–13. doi: 10.1136/thx.39.6.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Tsang KW, Chan K, Ho P, et al. Sputum elastase in steady-state bronchiectasis. Chest. 2000;117:420–6. doi: 10.1378/chest.117.2.420. [DOI] [PubMed] [Google Scholar]
  • 44.Lloberes P, Montserrat E, Montserrat JM, et al. Sputum sol phase proteins and elastase activity in patients with clinically stable bronchiectasis. Thorax. 1992;47:88–92. doi: 10.1136/thx.47.2.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Stevens T, Ekholm K, Gränse M, et al. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase. J Pharmacol Exp Ther. 2001;339:313–20. doi: 10.1124/jpet.111.182139. [DOI] [PubMed] [Google Scholar]
  • 46.Wang SM, Lei HY, Huang MC, et al. Modulation of cytokine production by intravenous immunoglobulin in patients with enterovirus 71-associated brainstem encephalitis. J Clin Virol. 2006;37:47–52. doi: 10.1016/j.jcv.2006.05.009. [DOI] [PubMed] [Google Scholar]
  • 47.Abe Y, Horiuchi A, Miyake M, et al. Anti-cytokine nature of natural human immunoglobulin: one possible mechanism of the clinical effect of intravenous immunoglobulin therapy. Immunol Rev. 1994;139:5–19. doi: 10.1111/j.1600-065x.1994.tb00854.x. [DOI] [PubMed] [Google Scholar]
  • 48.von Gunten S, Yousefi S, Seitz M, et al. Siglec-9 transduces apoptotic and non apoptotic death signals into neutrophils depending on the proinflammatory cytokine environment. Blood. 2005;106:1423–31. doi: 10.1182/blood-2004-10-4112. [DOI] [PubMed] [Google Scholar]
  • 49.Khatua B, Bhattacharya K, Mandal C. Sialoglycoproteins adsorbed by Pseudomonas aeruginosa facilitate their survival by impeding neutrophil extracellular trap through siglec-9. J Leukoc Biol. 2012;91:641–55. doi: 10.1189/jlb.0511260. [DOI] [PubMed] [Google Scholar]
  • 50.Carlin AF, Uchiyama S, Chang YC, et al. Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response. Blood. 2009;113:3333–6. doi: 10.1182/blood-2008-11-187302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Zhang JQ, Nicoll G, Jones C, et al. Siglec-9, a novel sialic acid binding member of the immunoglobulin superfamily expressed broadly on human blood leukocytes. J Biol Chem. 2000;275:22121–6. doi: 10.1074/jbc.M002788200. [DOI] [PubMed] [Google Scholar]
  • 52.Crocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nat Rev Immunol. 2007;7:255–66. doi: 10.1038/nri2056. [DOI] [PubMed] [Google Scholar]
  • 53.Avril T, Floyd H, Lopez F, et al. The membrane-proximal immunoreceptor tyrosine-based inhibitory motif is critical for the inhibitory signaling mediated by Siglecs-7 and -9, CD33-related Siglecs expressed on human monocytes and NK cells. J Immunol. 2004;173:6841–9. doi: 10.4049/jimmunol.173.11.6841. [DOI] [PubMed] [Google Scholar]
  • 54.Kustiawan I, Derksen N, Rispens T. Inactivated pepsin inhibits neutrophil activation by Fcgamma-receptor-dependent and independent stimuli. Clin Immunol. 2016;169:85–8. doi: 10.1016/j.clim.2016.06.008. [DOI] [PubMed] [Google Scholar]

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