Immunol Res (2014) 60:69–76 DOI 10.1007/s12026-014-8555-7
Defective functions of polymorphonuclear neutrophils in patients with common variable immunodeficiency Sarah Casulli • He´le`ne Coignard-Biehler • Karima Amazzough Michka Shoai-Tehrani • Jagadeesh Bayry • Nizar Mahlaoui • Carole Elbim • Srini V. Kaveri
•
Published online: 1 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Common variable immunodeficiency (CVID) is a heterogeneous antibody deficiency condition with alterations in T cell regulation and function, dendritic and B-cell compartment and represents the most frequent cause of symptomatic primary immunodeficiency. We addressed whether CVID is associated with abnormalities in the polymorphonuclear neutrophil (PMN) compartment, an important component of innate immunity and plays a key role in host defenses against invading microorganisms. We used flow cytometry to examine PMN phenotypic and functional abnormalities in CVID patients, using wholeblood conditions in order to avoid artifacts due to isolation procedures. We demonstrated that PMN from CVID patients displays, at resting state, a decreased expression of CD15, CD11b and CD16b, which might be related to an abnormality in neutrophil maturation. In addition, these neutrophils exhibit a decrease in degranulation,
Carole Elbim and Srini V. Kaveri have contributed equally to this work.
phagocytosis and reactive oxygen species production, as well as an increased death by apoptosis. These PMN abnormalities observed in CVID patients could result in an increased risk for recurrent bacterial infections. Keywords CVID Neutrophil Immunodeficiency Flow cytometry
Introduction Polymorphonuclear neutrophils (PMN) are the most important leukocytes of the innate immune system and are the first line of defense against bacterial and fungal pathogens. In response to pathogens, PMN rapidly migrate from blood to inflamed tissues via a multistep process of adhesive and migratory events. At the inflammatory site, PMN activation triggers the release of proteolytic enzymes and antimicrobial peptides, and the rapid production of reactive oxygen species (ROS), in the so-called oxidative
S. Casulli C. Elbim (&) UMR-S CR7, Sorbonne University, UPMC University Paris 06, 75005 Paris, France e-mail: [email protected]
J. Bayry S. V. Kaveri Centre de Recherche des Cordeliers, Equipe- Immunopathology and therapeutic immunointervention, Universite´ Pierre et Marie Curie – Paris 6, UMR S 1138, 15 rue de l’Ecole de Me´dicine, 75006 Paris, France
S. Casulli C. Elbim Centre d’Immunologie et des Maladies Infectieuses, UMR-S CR7, INSERM U1135, INSERM, 75013 Paris, France
J. Bayry S. V. Kaveri Universite´ Paris Descartes, UMR S 1138, 75006 Paris, France
H. Coignard-Biehler K. Amazzough M. Shoai-Tehrani Service des maladies infectieuses et tropicales, AP-HP, Hoˆpital Necker-Enfants Malades, 75015 Paris, France
N. Mahlaoui Centre de Re´fe´rence De´ficits Immunitaires He´re´ditaires (CEREDIH), AP-HP, Hoˆpital Universitaire Necker-Enfants Malades, 75015 Paris, France
J. Bayry S. V. Kaveri (&) Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 1138, 75006 Paris, France e-mail: [email protected]
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burst [1]. PMN are usually short-lived immune cells that die spontaneously by necrosis or apoptosis [1]. During microbial infections, circulating microbial products and endogenous pro-inflammatory mediators favor PMN survival, a mechanism critical for their tissue accumulation and effectiveness against pathogens [2, 3]. PMN disorders are involved in primary immunodeficiencies (PIDs) predisposing patients to recurrent bacterial infections. Several PMN defects have been identified in PIDs such as chronic granulomatous disease (CGD) [4], warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome [5] and leukocyte adhesion deficiency syndromes (LAD) [6]. CGD, an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any one of four genes encoding subunits of the phagocyte NADPH oxidase [4]. WHIM is caused by mutations of CXCR4 and is characterized by deficiency of neutrophils in circulation and by increased accumulation of mature neutrophils in the bone marrow [5]. LAD syndromes are primary immunodeficiency disorders that are classified as defects in adhesion-dependent functions of myeloid phagocytes, principally PMN and monocytes [6]. Further, a neutropenia is also observed in about 70 % of the patients with X-linked hyperimmunoglobulin M (IgM) syndrome (XHIGM) which is a rare immunodeficiency with normal or elevated serum IgM, reduced levels of IgG, IgA and IgE and defective T cell functions. The syndrome is due to mutations of the gene encoding for CD40L on chromosome X [7]. However, the role of PMN in common variable immunodeficiency (CVID) has not been elucidated. Common variable immunodeficiency is a heterogeneous antibody deficiency syndrome associated with an increased susceptibility to infections [8, 9]. CVID patients exhibit multiple defects of the immune system characterized by low levels of serum IgG, IgA and/or IgM, with a loss of antibody production. Defects in both, T and B cells, have been observed in some of these patients. T cell abnormalities, in particular a defective thymopoiesis of naı¨ve T cells, an increase in the percentage of activated T cells, an enhanced rate of apoptosis, abnormal T cell receptor signaling and cytokine production are observed in a large proportion of patients with CVID [10, 11]. The T cell dysfunctions involving defects in T and B-cell interactions could explain the defect in B-cell activation and differentiation leading to low immunoglobulin production observed in patients with CVID. Several abnormalities of innate immune system are also reported in these patients, notably a perturbed differentiation and maturation of dendritic cells (DC) [12, 13], an enhanced oxidative stress and chronic hyperactivity of monocytes [14, 15], a defective phagocytosis by monocytes [16], reduced absolute
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numbers of NK cells [17] and also neutropenia [18–20]. However, phenotype and functions of PMNs in these patients have not been addressed. Therefore, here we evaluated PMN functions and survival in CVID patients.
Materials and methods Patients and ethics statement Heparinized blood samples were collected from seven CVID patients, aged 28–73 years, at least 21 days following the last infusion of intravenous immunoglobulin (IVIg) (Table 1). As control, blood samples were from ten healthy donors, aged 21–62 years, collected at the Etablissement Franc¸ais du Sang (EFS, Pitie´ Salpeˆtrie`re Hospital, Paris, France) after ethical approval for the use of such material by the institutional review committees of INSERM and EFS (convention 12/EFS/079). The study was approved by the local institutional ethics committee (Comite´ de Protection des Personnes of Necker-Enfants Malades Hospital). After written informed consent had been obtained from the patients and the controls, whole blood was sampled, kept on ice and transported immediately to the laboratory. Determination of surface molecule expression on PMN Heparinized whole-blood samples were either kept on ice in order to analyze the expression of surface molecules at the surface of resting PMN (basal state), or incubated in a water bath for 45 min with PBS, Pam3CSK4 (TLR1/2 ligand, 1 lg/ml, Invivogen), LPS from E. coli serotype R515 (TLR4 ligand, 10 ng/ml; Invivogen, San Diego, CA), or TNF-a (5 ng/ml, R&D Systems) in order to evaluate the response of PMN to stimuli. Samples were then stained at 4 °C for 30 min with allophycocyanin (APC)-conjugated anti-human CD62L (clone DREG-56, BD Biosciences, San Jose, CA), phycoerythrin (PE)-conjugated anti-human CD11b (clone 2LPM19c, Dakopatts, Glostrup, Denmark), pacific blue (PB)-conjugated anti-CD15 (Beckman Coulter) and fluorescein isothiocyanate (FITC)-conjugated antihuman CD16b antibodies (clone 1D3, Beckman Coulter). Erythrocytes were lysed with FACS lysing solution, and white blood cells were resuspended in 1X BD Cell Fix and analyzed by flow cytometry. Nonspecific Ab binding was determined on cells incubated with the same concentration of an irrelevant Ab of the same isotype. Measurement of phagocytosis The percentage of PMN phagocytosing opsonized E. coli as well as the mean fluorescence intensity (MFI) was
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Table 1 Characteristics of CVID patients CVID patients (n = 7)
Age (years)
Age at onset (years)
Sex
1 2
28
11
53
35
3
56
4 5
Clinical and laboratory findings
Other features and associated complications
Plasma IgG (mg/ml)
IVIg treatment duration (years)
CRP (mg/l)
Neutrophil count (cells/mm3)
F
10, 2
[10
11
6,200
Bronchiectasis, weight loss
M
9, 8
[10
23
4,400
Idiopathic thrombocytopenic purpura, lymphoid hyperplasia, thyroid dysfunction
44
F
11, 1
12
6
4,300
Addison’s Diseases, Biermer’s anaemia, Hashimoto’s thyroiditis, adrenal insufficiency, bronchiectasis, inflammation of the digestive tract
50
10
M
6,0
73
22
F
12, 2
6
62
38
F
7
69
65
F
1
15
6,800
Inflammation of the digestive tract
[10
6
1,900
Bronchiectasis, high blood pressure
9, 2
24
7
900
10,4
4
6
1,800
Idiopathic thrombocytopenic purpura, nodular regenerative hyperplasia, high blood pressure Peripheral neuropathy
F female; M male The Table summarizes the age of the patients during sampling and during the onset of the disease, sex and clinical parameters such as the level of plasma IgG trough level at the time of study, CRP level, PMN count and associated complications such as intestinal inflammatory disease, organspecific autoimmunity, lymphoid hyperplasia
measured in whole blood by using the Phagotest Kit (Glycotope Biotechnology). Whole-blood samples (100 ll) were incubated with opsonized fluorescein (FITC)-conjugated E. coli bacteria (2 9 107 per 20 ll) for 10 min in a water bath at 37 °C, the phagocytosis is then stopped in ice and erythrocytes are lysed before analysis by flow cytometry. Measurement of the PMN oxidative burst Superoxide anion (O2-) production was measured with a flow cytometric assay derived from the hydroethidine (HE) oxidation technique [21]. Heparin whole-blood samples (500 ll) were loaded for 15 min with 1,500 ng/ml HE (Fluka, Buchs, Switzerland) at 37 °C, and then incubated for 45 min with PBS or various stimuli, as described above. Samples were then treated with PBS or 10-6 M fMLP (Sigma Chemical CO., St Louis, MO) for 5 min. Red cells were lysed as described above, and white cells were resuspended in 1X BD Cell Fix. Measurement of PMN apoptosis Apoptosis of PMN in whole blood was quantified by using annexin V and the impermeant nuclear dye 7-amino-actinomycin D (7-AAD) as previously described [22]. PMN apoptosis was measured after incubation in 24-well tissue culture plates, for 20 h, at 37 °C. Whole-blood samples (100 ll) were then washed twice in PBS, incubated with APC-anti-CD15 (clone HI98, BD Biosciences) for 15 min, and then incubated with FITC-annexin V for 15 min. After dilution in PBS
(500 ll), the samples were incubated with 7-AAD (BD Biosciences) at room temperature for 15 min and resuspended in 1X BD Cell Fix and analyzed by flow cytometry. Flow cytometry analysis Cells were acquired with the GalliosTM flow cytometer and analyzed with Kaluza software. To determine the expression of surface molecules on PMN, and ROS production by PMNs, forward and side scatter characteristics were used to identify the PMN population and to gate out other cells and debris, and 10,000 events were counted per sample. Results were expressed as MFI. To measure PMN phagocytosis, a ‘‘live’’ gate on leukocyte is set during data acquisition allowing excluding of bacteria. A total of 10,000–15,000 leukocytes were collected per sample and the granulocyte cluster is gated in the software program in the scatter diagram (lin FSC vs. lin SSC). The MFI of FITC correlates with the number of bacteria per individual leukocyte. Data were expressed as the PMN phagocytic index: % phagocytic PMN 9 MFI. To measure cell death in whole blood by flow cytometry, PMNs were identified as CD15high cells and 2 9 105 events were counted per sample. Results were expressed as the percentage of total apoptotic cells. Statistical analysis Data are reported as mean ± SEM. Comparisons were based on Mann–Whitney U test, using Prism 3.0 software (GraphPad Prism 5 software). The significance level was set at p \ 0.05.
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Fig. 1 Polymorphonuclear neutrophils from CVID patients display decreased expression of cell-surface markers. Expression of cell-surface molecules on resting PMN was studied on whole-blood samples maintained at 4 °C and stained with specific mAbs before analysis by flow cytometry. The surface expression of CD11b (a), CD62L (b), CD16b (c) and CD15 (d) on resting PMN is indicated for 7 CVID patients (open diamond) as compared to 10 healthy donors controls (filled circles). Results are expressed as mean fluorescence intensity. A horizontal bar for each marker indicates the mean values. Statistical significance as determined by the nonparametric Mann–Whitney test is indicated *p \ 0.05, **p \ 0.01, ***p \ 0.001
Results and discussion At resting state, PMN of CVID patients expressed markedly reduced levels of CD11b adhesion molecule on their surface (20.2 ± 1.4) as compared with PMN of healthy controls (33 ± 1.5) (Fig. 1a). PMN from CVID patients expressed normal levels of CD62L (Fig. 1b) comparable to those of healthy donors. The expression of CD16b (FccRIII) and CD15 on PMN surface from CVID patients was significantly lower than in controls (Fig. 1c, d). CD62L is an adhesion molecule constitutively expressed on the surface of PMN. By contrast, CD11b, a subunit of the integrin CD11b/CD18 (complement receptor 3) is present mostly within a specific granule subset in PMN and is degranulated following PMN activation. The CD11b/ CD18 is important for proper functioning of PMN, facilitating the phagocytosis of complement protein (C3b)coated particles and mediating adhesive interactions of PMN with endothelial cells and extracellular matrix proteins [23]. CD16b is a glycosyl phosphatidyl (GPI)anchored low-affinity IgG receptor (FccRIII) exclusively expressed on human PMN; CD16b cross-linking induces the effector functions such as degranulation and activation of respiratory burst [24]. Maturation of PMN into the neutrophilic pathway is indicated by the acquisition of the CD15 antigen, followed by CD11b and CD16b in that order. The decrease in the MFI of CD15, CD11b and CD16b suggests a deficiency in the maturation of PMN in CVID patients. It is possible that
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PMN from CVID patients, released into the peripheral circulation from the bone marrow, are deficient in these cytoplasmic granule proteins. These results are indeed in agreement with an altered granulopoiesis observed in CVID [25]. As previously reported in X-linked hyperIgM syndrome [26], a possible defective CD40-CD40L interaction might explain the observed alteration in granulopoiesis in CVID patients. Bone marrow stromal cells express CD40 on their surface and upon engagement with CD40L, they upregulate the expression of GM-CSF and G-CSF, two of the key regulators of granulopoiesis [7]. More than 80 % of CVID patients show severely reduced switched memory B-cells indicative of a defective germinal center development as found in CD40L deficiency [27]. Nevertheless, contradictory results have been reported regarding the expression of CD40/CD40L on various immune cells in CVID patients [28–31] and the precise role of CD40L defect in the pathophysiology of the disease remains to be elucidated. Finally, an impaired PMN maturation in CVID patients might be related to altered expression of chemokine receptors which are key parameters in this setting [32–34]. Retention and release of mature PMN is determined by the balance between the two chemokine receptors, CXCR4, favoring retention and CXCR2, aiding the release [35]. Thus, measurement of the expression of these two chemokine receptors in CVID patients warrants further exploration. A defective maturation of several others immune cells, in particular DC and B-cells, has been reported in CVID
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Fig. 2 Polymorphonuclear neutrophils from CVID patients display impaired functional properties. a Whole-blood samples were incubated with fluorescent opsonized E. coli bacteria for 10 min in a water bath at 37 °C. After gating on granulocytes (FSC/SCC), PMN phagocytosis was analyzed and expressed as the PMN phagocytic index. Results are indicated for 7 CVID patients (open diamond) as compared to 10 healthy donors controls (filled circles). b and c Expression of adhesion molecules on PMN. CD62L (b) and CD11b (c) expression on PMN surface were measured after incubation of whole-blood samples at 37 °C for 45 min with PBS, Pam3CSK4
(TLR1/2 agonist, 1 lg/mL), LPS (TLR4 agonist), or TNF-a (TNF). d ROS production by PMNs was measured after loading with HE, pretreatment of whole-blood samples for 45 min with PBS, TLR1/2 agonist TLR4 agonist or TNF and stimulation for 5 min with fMLP. All measurements were taken in healthy controls (open bars) and CVID patients (gray bars). Results are expressed as mean fluorescence intensity and values are mean ± SEM. Statistical significance as determined by the nonparametric Mann–Whitney test is indicated *p \ 0.05, **p \ 0.01, ***p \ 0.001
patients. In fact, DCs from patients with CVID have been shown to have reduced expression of surface molecules associated with maturation, such as CD40, that could compromise antigen presentation [12, 36]. DCs from CVID patients display severely perturbed differentiation, maturation, and function and express markedly reduced levels of the costimulatory molecules that are critical for
T cell stimulation [12, 37]. Patients with CVID also present the phenotype of defective B lymphocytes related to impaired B-cell maturation and defects in memory B-cell differentiation [38] as well as thymic dysregulation [39, 40]. The uniform nature of the defects of neutrophils is reminder of those previously observed with DC and B cells [12].
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Fig. 3 Polymorphonuclear neutrophils from CVID patients display an increased in spontaneous apoptosis. Wholeblood samples were incubated with PBS in 24-well tissue cultures plates at 37 °C with 5 % CO2 for 24 h. Samples were then incubated at 4 °C for 15 min with FITC-anti-CD15 Ab and stained with annexin V and 7-AAD as described in ‘‘Materials and methods’’ section. The fluorescence of anti-CD15 Ab was used to identify PMN as CD15? cells and to gate out other cells, erythrocytes and debris. Fluorescence analysis was performed on this gated cell population (a). All measurements were taken in healthy controls (filled circles) and CVID patients (open diamond). Results are expressed as the percentage of total apoptotic cells (early and late apoptotic cells) (b). Values are mean ± SEM. ***p \ 0.05
The phenotypic alterations observed in CVID patients might be associated with functional abnormalities. PMN with abnormal CD11b/CD18 and CD16b phenotype displays functional alterations that include decreased phagocytosis and oxidative burst [41–44]. Phagocytosis is a crucial part of host defense against invading extracellular microorganisms. The ingestion of bacteria by phagocytes involves a variety of cell membrane recognition structures and is generally dependent on the proper opsonization of the microorganisms, in particular by C3 complement molecules and immunoglobulins (Ig). Given that CVID patients exhibit Ig deficit, we studied the intrinsic capacity of PMN phagocytosis using opsonized bacteria. We observed that phagocytic index was significantly lower in PMN from CVID patients compared with controls (Fig. 2a) that reflected an intrinsic defect in the capacity of PMN phagocytosis. We also analyzed the PMN functional capacity in response to different stimuli. Following stimulation with TLR4, TLR1/2 ligands and TNF, PMN from CVID patients exhibited a normal CD62L shedding (Fig. 2c), whereas the up-regulation of CD11b/CD18 (Fig. 2b) was defective. In a similar manner, we observed that the ROS production induced by pro-inflammatory stimuli was lower in CVID patients than in healthy donors (Fig. 2d). It would thus be of interest to investigate whether the reduction in the expression of CD11b and in respiratory burst is secondary to reduced amount of total cellular CD11b and a reduction
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in the amount of NADPH oxidase component, in particular of p22/gp91 phox or whether this is due to a reduced capacity to mobilize these molecules to the surface membrane. PMN are usually short-lived immune cells, which die spontaneously mainly by apoptosis. Aged PMN undergo spontaneous apoptosis in the absence of cytokines or other pro-inflammatory agents, prior to their removal by macrophages [1]. This phagocytic removal of intact, apoptotic neutrophils prevents them from releasing their cytotoxic content into the extracellular environment that would occur if the cells died by necrosis. However, shortened PMN survival due to increased apoptosis has been involved in the increased susceptibility to severe and recurrent infections in different pathological situations [45]. PMN death by apoptosis is increased in patients with CVID than in healthy donors (Fig. 3), which may account for enhanced recurrent infections. This alteration could be due to abnormalities in signaling pathway, especially death receptor pathway or polyclonal expansion of large granular lymphocytes [19, 46]. Although as discussed above, other cellular anomalies are well defined, CVID is generally considered as a B-lymphocyte defect. It is, however, not clear whether the observed PMN abnormalities are consequences of inherent neutrophil defects caused by the same genetic defects as those that cause deficiencies in B-cell functions. Genetic defects in TACI (TNFRST13B), ICOS, BAFF-R and CD19
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account only for a minority (15–20 %) of cases of CVID; the molecular pathophysiology of the remaining cases remains undefined and could be polygenic. In summary, CVID patients exhibit defects in a wide range of immunological functions including those related to B-cell numbers and functions, DC maturation, NKT cells, Th17 cells [10, 12, 13, 47, 48]. Our study showed that PMN from CVID patients displays a defective phenotype with decreased functional capacities and survival, and thereby may account for enhanced recurrent infections. Multiple defects in the immune system, including defective PMN function, thus appear to be prominent features of CVID. IVIg at low doses has been previously reported to induce in vitro PMN activation associated with a prolonged survival [49]. In addition to primary and secondary immune deficiency, IVIg is used in a large number of autoimmune and inflammatory diseases [50–54]. Indeed, in CVID patients, IVIg has been shown to modulate the functions of different cells of both innate and adaptive compartments [55, 56]. Thus, it would be highly interesting to examine the PMN survival and functions following IVIg administration in CVID patients. Acknowledgments Supported by Institut National de la Sante´ et de la Recherche Me´dicale, Universite´ Pierre et Marie Curie, Universite´ Paris Descartes (S.V.K. and J.B.), Centre National de la Recherche Scientifique (S.V.K.).
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Defective functions of polymorphonuclear neutrophils in patients with common variable immunodeficiency Sarah Casulli • He´le`ne Coignard-Biehler • Karima Amazzough Michka Shoai-Tehrani • Jagadeesh Bayry • Nizar Mahlaoui • Carole Elbim • Srini V. Kaveri
•
Published online: 1 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Common variable immunodeficiency (CVID) is a heterogeneous antibody deficiency condition with alterations in T cell regulation and function, dendritic and B-cell compartment and represents the most frequent cause of symptomatic primary immunodeficiency. We addressed whether CVID is associated with abnormalities in the polymorphonuclear neutrophil (PMN) compartment, an important component of innate immunity and plays a key role in host defenses against invading microorganisms. We used flow cytometry to examine PMN phenotypic and functional abnormalities in CVID patients, using wholeblood conditions in order to avoid artifacts due to isolation procedures. We demonstrated that PMN from CVID patients displays, at resting state, a decreased expression of CD15, CD11b and CD16b, which might be related to an abnormality in neutrophil maturation. In addition, these neutrophils exhibit a decrease in degranulation,
Carole Elbim and Srini V. Kaveri have contributed equally to this work.
phagocytosis and reactive oxygen species production, as well as an increased death by apoptosis. These PMN abnormalities observed in CVID patients could result in an increased risk for recurrent bacterial infections. Keywords CVID Neutrophil Immunodeficiency Flow cytometry
Introduction Polymorphonuclear neutrophils (PMN) are the most important leukocytes of the innate immune system and are the first line of defense against bacterial and fungal pathogens. In response to pathogens, PMN rapidly migrate from blood to inflamed tissues via a multistep process of adhesive and migratory events. At the inflammatory site, PMN activation triggers the release of proteolytic enzymes and antimicrobial peptides, and the rapid production of reactive oxygen species (ROS), in the so-called oxidative
S. Casulli C. Elbim (&) UMR-S CR7, Sorbonne University, UPMC University Paris 06, 75005 Paris, France e-mail: [email protected]
J. Bayry S. V. Kaveri Centre de Recherche des Cordeliers, Equipe- Immunopathology and therapeutic immunointervention, Universite´ Pierre et Marie Curie – Paris 6, UMR S 1138, 15 rue de l’Ecole de Me´dicine, 75006 Paris, France
S. Casulli C. Elbim Centre d’Immunologie et des Maladies Infectieuses, UMR-S CR7, INSERM U1135, INSERM, 75013 Paris, France
J. Bayry S. V. Kaveri Universite´ Paris Descartes, UMR S 1138, 75006 Paris, France
H. Coignard-Biehler K. Amazzough M. Shoai-Tehrani Service des maladies infectieuses et tropicales, AP-HP, Hoˆpital Necker-Enfants Malades, 75015 Paris, France
N. Mahlaoui Centre de Re´fe´rence De´ficits Immunitaires He´re´ditaires (CEREDIH), AP-HP, Hoˆpital Universitaire Necker-Enfants Malades, 75015 Paris, France
J. Bayry S. V. Kaveri (&) Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 1138, 75006 Paris, France e-mail: [email protected]
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burst [1]. PMN are usually short-lived immune cells that die spontaneously by necrosis or apoptosis [1]. During microbial infections, circulating microbial products and endogenous pro-inflammatory mediators favor PMN survival, a mechanism critical for their tissue accumulation and effectiveness against pathogens [2, 3]. PMN disorders are involved in primary immunodeficiencies (PIDs) predisposing patients to recurrent bacterial infections. Several PMN defects have been identified in PIDs such as chronic granulomatous disease (CGD) [4], warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome [5] and leukocyte adhesion deficiency syndromes (LAD) [6]. CGD, an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any one of four genes encoding subunits of the phagocyte NADPH oxidase [4]. WHIM is caused by mutations of CXCR4 and is characterized by deficiency of neutrophils in circulation and by increased accumulation of mature neutrophils in the bone marrow [5]. LAD syndromes are primary immunodeficiency disorders that are classified as defects in adhesion-dependent functions of myeloid phagocytes, principally PMN and monocytes [6]. Further, a neutropenia is also observed in about 70 % of the patients with X-linked hyperimmunoglobulin M (IgM) syndrome (XHIGM) which is a rare immunodeficiency with normal or elevated serum IgM, reduced levels of IgG, IgA and IgE and defective T cell functions. The syndrome is due to mutations of the gene encoding for CD40L on chromosome X [7]. However, the role of PMN in common variable immunodeficiency (CVID) has not been elucidated. Common variable immunodeficiency is a heterogeneous antibody deficiency syndrome associated with an increased susceptibility to infections [8, 9]. CVID patients exhibit multiple defects of the immune system characterized by low levels of serum IgG, IgA and/or IgM, with a loss of antibody production. Defects in both, T and B cells, have been observed in some of these patients. T cell abnormalities, in particular a defective thymopoiesis of naı¨ve T cells, an increase in the percentage of activated T cells, an enhanced rate of apoptosis, abnormal T cell receptor signaling and cytokine production are observed in a large proportion of patients with CVID [10, 11]. The T cell dysfunctions involving defects in T and B-cell interactions could explain the defect in B-cell activation and differentiation leading to low immunoglobulin production observed in patients with CVID. Several abnormalities of innate immune system are also reported in these patients, notably a perturbed differentiation and maturation of dendritic cells (DC) [12, 13], an enhanced oxidative stress and chronic hyperactivity of monocytes [14, 15], a defective phagocytosis by monocytes [16], reduced absolute
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numbers of NK cells [17] and also neutropenia [18–20]. However, phenotype and functions of PMNs in these patients have not been addressed. Therefore, here we evaluated PMN functions and survival in CVID patients.
Materials and methods Patients and ethics statement Heparinized blood samples were collected from seven CVID patients, aged 28–73 years, at least 21 days following the last infusion of intravenous immunoglobulin (IVIg) (Table 1). As control, blood samples were from ten healthy donors, aged 21–62 years, collected at the Etablissement Franc¸ais du Sang (EFS, Pitie´ Salpeˆtrie`re Hospital, Paris, France) after ethical approval for the use of such material by the institutional review committees of INSERM and EFS (convention 12/EFS/079). The study was approved by the local institutional ethics committee (Comite´ de Protection des Personnes of Necker-Enfants Malades Hospital). After written informed consent had been obtained from the patients and the controls, whole blood was sampled, kept on ice and transported immediately to the laboratory. Determination of surface molecule expression on PMN Heparinized whole-blood samples were either kept on ice in order to analyze the expression of surface molecules at the surface of resting PMN (basal state), or incubated in a water bath for 45 min with PBS, Pam3CSK4 (TLR1/2 ligand, 1 lg/ml, Invivogen), LPS from E. coli serotype R515 (TLR4 ligand, 10 ng/ml; Invivogen, San Diego, CA), or TNF-a (5 ng/ml, R&D Systems) in order to evaluate the response of PMN to stimuli. Samples were then stained at 4 °C for 30 min with allophycocyanin (APC)-conjugated anti-human CD62L (clone DREG-56, BD Biosciences, San Jose, CA), phycoerythrin (PE)-conjugated anti-human CD11b (clone 2LPM19c, Dakopatts, Glostrup, Denmark), pacific blue (PB)-conjugated anti-CD15 (Beckman Coulter) and fluorescein isothiocyanate (FITC)-conjugated antihuman CD16b antibodies (clone 1D3, Beckman Coulter). Erythrocytes were lysed with FACS lysing solution, and white blood cells were resuspended in 1X BD Cell Fix and analyzed by flow cytometry. Nonspecific Ab binding was determined on cells incubated with the same concentration of an irrelevant Ab of the same isotype. Measurement of phagocytosis The percentage of PMN phagocytosing opsonized E. coli as well as the mean fluorescence intensity (MFI) was
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Table 1 Characteristics of CVID patients CVID patients (n = 7)
Age (years)
Age at onset (years)
Sex
1 2
28
11
53
35
3
56
4 5
Clinical and laboratory findings
Other features and associated complications
Plasma IgG (mg/ml)
IVIg treatment duration (years)
CRP (mg/l)
Neutrophil count (cells/mm3)
F
10, 2
[10
11
6,200
Bronchiectasis, weight loss
M
9, 8
[10
23
4,400
Idiopathic thrombocytopenic purpura, lymphoid hyperplasia, thyroid dysfunction
44
F
11, 1
12
6
4,300
Addison’s Diseases, Biermer’s anaemia, Hashimoto’s thyroiditis, adrenal insufficiency, bronchiectasis, inflammation of the digestive tract
50
10
M
6,0
73
22
F
12, 2
6
62
38
F
7
69
65
F
1
15
6,800
Inflammation of the digestive tract
[10
6
1,900
Bronchiectasis, high blood pressure
9, 2
24
7
900
10,4
4
6
1,800
Idiopathic thrombocytopenic purpura, nodular regenerative hyperplasia, high blood pressure Peripheral neuropathy
F female; M male The Table summarizes the age of the patients during sampling and during the onset of the disease, sex and clinical parameters such as the level of plasma IgG trough level at the time of study, CRP level, PMN count and associated complications such as intestinal inflammatory disease, organspecific autoimmunity, lymphoid hyperplasia
measured in whole blood by using the Phagotest Kit (Glycotope Biotechnology). Whole-blood samples (100 ll) were incubated with opsonized fluorescein (FITC)-conjugated E. coli bacteria (2 9 107 per 20 ll) for 10 min in a water bath at 37 °C, the phagocytosis is then stopped in ice and erythrocytes are lysed before analysis by flow cytometry. Measurement of the PMN oxidative burst Superoxide anion (O2-) production was measured with a flow cytometric assay derived from the hydroethidine (HE) oxidation technique [21]. Heparin whole-blood samples (500 ll) were loaded for 15 min with 1,500 ng/ml HE (Fluka, Buchs, Switzerland) at 37 °C, and then incubated for 45 min with PBS or various stimuli, as described above. Samples were then treated with PBS or 10-6 M fMLP (Sigma Chemical CO., St Louis, MO) for 5 min. Red cells were lysed as described above, and white cells were resuspended in 1X BD Cell Fix. Measurement of PMN apoptosis Apoptosis of PMN in whole blood was quantified by using annexin V and the impermeant nuclear dye 7-amino-actinomycin D (7-AAD) as previously described [22]. PMN apoptosis was measured after incubation in 24-well tissue culture plates, for 20 h, at 37 °C. Whole-blood samples (100 ll) were then washed twice in PBS, incubated with APC-anti-CD15 (clone HI98, BD Biosciences) for 15 min, and then incubated with FITC-annexin V for 15 min. After dilution in PBS
(500 ll), the samples were incubated with 7-AAD (BD Biosciences) at room temperature for 15 min and resuspended in 1X BD Cell Fix and analyzed by flow cytometry. Flow cytometry analysis Cells were acquired with the GalliosTM flow cytometer and analyzed with Kaluza software. To determine the expression of surface molecules on PMN, and ROS production by PMNs, forward and side scatter characteristics were used to identify the PMN population and to gate out other cells and debris, and 10,000 events were counted per sample. Results were expressed as MFI. To measure PMN phagocytosis, a ‘‘live’’ gate on leukocyte is set during data acquisition allowing excluding of bacteria. A total of 10,000–15,000 leukocytes were collected per sample and the granulocyte cluster is gated in the software program in the scatter diagram (lin FSC vs. lin SSC). The MFI of FITC correlates with the number of bacteria per individual leukocyte. Data were expressed as the PMN phagocytic index: % phagocytic PMN 9 MFI. To measure cell death in whole blood by flow cytometry, PMNs were identified as CD15high cells and 2 9 105 events were counted per sample. Results were expressed as the percentage of total apoptotic cells. Statistical analysis Data are reported as mean ± SEM. Comparisons were based on Mann–Whitney U test, using Prism 3.0 software (GraphPad Prism 5 software). The significance level was set at p \ 0.05.
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Fig. 1 Polymorphonuclear neutrophils from CVID patients display decreased expression of cell-surface markers. Expression of cell-surface molecules on resting PMN was studied on whole-blood samples maintained at 4 °C and stained with specific mAbs before analysis by flow cytometry. The surface expression of CD11b (a), CD62L (b), CD16b (c) and CD15 (d) on resting PMN is indicated for 7 CVID patients (open diamond) as compared to 10 healthy donors controls (filled circles). Results are expressed as mean fluorescence intensity. A horizontal bar for each marker indicates the mean values. Statistical significance as determined by the nonparametric Mann–Whitney test is indicated *p \ 0.05, **p \ 0.01, ***p \ 0.001
Results and discussion At resting state, PMN of CVID patients expressed markedly reduced levels of CD11b adhesion molecule on their surface (20.2 ± 1.4) as compared with PMN of healthy controls (33 ± 1.5) (Fig. 1a). PMN from CVID patients expressed normal levels of CD62L (Fig. 1b) comparable to those of healthy donors. The expression of CD16b (FccRIII) and CD15 on PMN surface from CVID patients was significantly lower than in controls (Fig. 1c, d). CD62L is an adhesion molecule constitutively expressed on the surface of PMN. By contrast, CD11b, a subunit of the integrin CD11b/CD18 (complement receptor 3) is present mostly within a specific granule subset in PMN and is degranulated following PMN activation. The CD11b/ CD18 is important for proper functioning of PMN, facilitating the phagocytosis of complement protein (C3b)coated particles and mediating adhesive interactions of PMN with endothelial cells and extracellular matrix proteins [23]. CD16b is a glycosyl phosphatidyl (GPI)anchored low-affinity IgG receptor (FccRIII) exclusively expressed on human PMN; CD16b cross-linking induces the effector functions such as degranulation and activation of respiratory burst [24]. Maturation of PMN into the neutrophilic pathway is indicated by the acquisition of the CD15 antigen, followed by CD11b and CD16b in that order. The decrease in the MFI of CD15, CD11b and CD16b suggests a deficiency in the maturation of PMN in CVID patients. It is possible that
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PMN from CVID patients, released into the peripheral circulation from the bone marrow, are deficient in these cytoplasmic granule proteins. These results are indeed in agreement with an altered granulopoiesis observed in CVID [25]. As previously reported in X-linked hyperIgM syndrome [26], a possible defective CD40-CD40L interaction might explain the observed alteration in granulopoiesis in CVID patients. Bone marrow stromal cells express CD40 on their surface and upon engagement with CD40L, they upregulate the expression of GM-CSF and G-CSF, two of the key regulators of granulopoiesis [7]. More than 80 % of CVID patients show severely reduced switched memory B-cells indicative of a defective germinal center development as found in CD40L deficiency [27]. Nevertheless, contradictory results have been reported regarding the expression of CD40/CD40L on various immune cells in CVID patients [28–31] and the precise role of CD40L defect in the pathophysiology of the disease remains to be elucidated. Finally, an impaired PMN maturation in CVID patients might be related to altered expression of chemokine receptors which are key parameters in this setting [32–34]. Retention and release of mature PMN is determined by the balance between the two chemokine receptors, CXCR4, favoring retention and CXCR2, aiding the release [35]. Thus, measurement of the expression of these two chemokine receptors in CVID patients warrants further exploration. A defective maturation of several others immune cells, in particular DC and B-cells, has been reported in CVID
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Fig. 2 Polymorphonuclear neutrophils from CVID patients display impaired functional properties. a Whole-blood samples were incubated with fluorescent opsonized E. coli bacteria for 10 min in a water bath at 37 °C. After gating on granulocytes (FSC/SCC), PMN phagocytosis was analyzed and expressed as the PMN phagocytic index. Results are indicated for 7 CVID patients (open diamond) as compared to 10 healthy donors controls (filled circles). b and c Expression of adhesion molecules on PMN. CD62L (b) and CD11b (c) expression on PMN surface were measured after incubation of whole-blood samples at 37 °C for 45 min with PBS, Pam3CSK4
(TLR1/2 agonist, 1 lg/mL), LPS (TLR4 agonist), or TNF-a (TNF). d ROS production by PMNs was measured after loading with HE, pretreatment of whole-blood samples for 45 min with PBS, TLR1/2 agonist TLR4 agonist or TNF and stimulation for 5 min with fMLP. All measurements were taken in healthy controls (open bars) and CVID patients (gray bars). Results are expressed as mean fluorescence intensity and values are mean ± SEM. Statistical significance as determined by the nonparametric Mann–Whitney test is indicated *p \ 0.05, **p \ 0.01, ***p \ 0.001
patients. In fact, DCs from patients with CVID have been shown to have reduced expression of surface molecules associated with maturation, such as CD40, that could compromise antigen presentation [12, 36]. DCs from CVID patients display severely perturbed differentiation, maturation, and function and express markedly reduced levels of the costimulatory molecules that are critical for
T cell stimulation [12, 37]. Patients with CVID also present the phenotype of defective B lymphocytes related to impaired B-cell maturation and defects in memory B-cell differentiation [38] as well as thymic dysregulation [39, 40]. The uniform nature of the defects of neutrophils is reminder of those previously observed with DC and B cells [12].
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Fig. 3 Polymorphonuclear neutrophils from CVID patients display an increased in spontaneous apoptosis. Wholeblood samples were incubated with PBS in 24-well tissue cultures plates at 37 °C with 5 % CO2 for 24 h. Samples were then incubated at 4 °C for 15 min with FITC-anti-CD15 Ab and stained with annexin V and 7-AAD as described in ‘‘Materials and methods’’ section. The fluorescence of anti-CD15 Ab was used to identify PMN as CD15? cells and to gate out other cells, erythrocytes and debris. Fluorescence analysis was performed on this gated cell population (a). All measurements were taken in healthy controls (filled circles) and CVID patients (open diamond). Results are expressed as the percentage of total apoptotic cells (early and late apoptotic cells) (b). Values are mean ± SEM. ***p \ 0.05
The phenotypic alterations observed in CVID patients might be associated with functional abnormalities. PMN with abnormal CD11b/CD18 and CD16b phenotype displays functional alterations that include decreased phagocytosis and oxidative burst [41–44]. Phagocytosis is a crucial part of host defense against invading extracellular microorganisms. The ingestion of bacteria by phagocytes involves a variety of cell membrane recognition structures and is generally dependent on the proper opsonization of the microorganisms, in particular by C3 complement molecules and immunoglobulins (Ig). Given that CVID patients exhibit Ig deficit, we studied the intrinsic capacity of PMN phagocytosis using opsonized bacteria. We observed that phagocytic index was significantly lower in PMN from CVID patients compared with controls (Fig. 2a) that reflected an intrinsic defect in the capacity of PMN phagocytosis. We also analyzed the PMN functional capacity in response to different stimuli. Following stimulation with TLR4, TLR1/2 ligands and TNF, PMN from CVID patients exhibited a normal CD62L shedding (Fig. 2c), whereas the up-regulation of CD11b/CD18 (Fig. 2b) was defective. In a similar manner, we observed that the ROS production induced by pro-inflammatory stimuli was lower in CVID patients than in healthy donors (Fig. 2d). It would thus be of interest to investigate whether the reduction in the expression of CD11b and in respiratory burst is secondary to reduced amount of total cellular CD11b and a reduction
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in the amount of NADPH oxidase component, in particular of p22/gp91 phox or whether this is due to a reduced capacity to mobilize these molecules to the surface membrane. PMN are usually short-lived immune cells, which die spontaneously mainly by apoptosis. Aged PMN undergo spontaneous apoptosis in the absence of cytokines or other pro-inflammatory agents, prior to their removal by macrophages [1]. This phagocytic removal of intact, apoptotic neutrophils prevents them from releasing their cytotoxic content into the extracellular environment that would occur if the cells died by necrosis. However, shortened PMN survival due to increased apoptosis has been involved in the increased susceptibility to severe and recurrent infections in different pathological situations [45]. PMN death by apoptosis is increased in patients with CVID than in healthy donors (Fig. 3), which may account for enhanced recurrent infections. This alteration could be due to abnormalities in signaling pathway, especially death receptor pathway or polyclonal expansion of large granular lymphocytes [19, 46]. Although as discussed above, other cellular anomalies are well defined, CVID is generally considered as a B-lymphocyte defect. It is, however, not clear whether the observed PMN abnormalities are consequences of inherent neutrophil defects caused by the same genetic defects as those that cause deficiencies in B-cell functions. Genetic defects in TACI (TNFRST13B), ICOS, BAFF-R and CD19
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account only for a minority (15–20 %) of cases of CVID; the molecular pathophysiology of the remaining cases remains undefined and could be polygenic. In summary, CVID patients exhibit defects in a wide range of immunological functions including those related to B-cell numbers and functions, DC maturation, NKT cells, Th17 cells [10, 12, 13, 47, 48]. Our study showed that PMN from CVID patients displays a defective phenotype with decreased functional capacities and survival, and thereby may account for enhanced recurrent infections. Multiple defects in the immune system, including defective PMN function, thus appear to be prominent features of CVID. IVIg at low doses has been previously reported to induce in vitro PMN activation associated with a prolonged survival [49]. In addition to primary and secondary immune deficiency, IVIg is used in a large number of autoimmune and inflammatory diseases [50–54]. Indeed, in CVID patients, IVIg has been shown to modulate the functions of different cells of both innate and adaptive compartments [55, 56]. Thus, it would be highly interesting to examine the PMN survival and functions following IVIg administration in CVID patients. Acknowledgments Supported by Institut National de la Sante´ et de la Recherche Me´dicale, Universite´ Pierre et Marie Curie, Universite´ Paris Descartes (S.V.K. and J.B.), Centre National de la Recherche Scientifique (S.V.K.).
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