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Preparations of intravenous immunoglobulins diminishthe number and proinflammatory response ofCD14+CD16++ monocytes in common variableimmunodeficiency (CVID) patientsMaciej Siedlar a,⁎, Magdalena Strachb, Karolina Bukowska-Strakovaa,Marzena Lenart a, Anna Szaflarskaa, Kazimierz Węglarczyka,Magdalena Rutkowskaa, Monika Baj-Krzyworzekaa,Anna Pituch-Noworolska a, Danuta Kowalczyka, Tomasz Grodzicki b,Loems Ziegler-Heitbrock c,d, Marek Zembala a

a Department of Clinical Immunology, Polish-American Institute of Pediatrics, Jagiellonian University Medical College,Krakow, Polandb Department of InternalMedicine andGerontology, UniversityHospital, JagiellonianUniversityMedical College, Krakow,Polandc Asklepios-Fachklinik and Helmholtz Zentrum München, German Research Center for Environmental Health, Gauting, Germanyd Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK

Received 24 May 2010; accepted with revision 8 January 2011Available online 15 January 2011

KEYWORDSCD14+CD16++ monocytes;Intravenousimmunoglobulins;CD32B;Common variableimmunodeficiency

Abstract We have studied the effect of intravenous immunoglobulins (IVIG) on monocytesubpopulations and cytokine production in patients with CVID. The absolute number ofCD14+CD16++ monocytes decreased on average 2.5-fold 4 h after IVIG and after 20 h returnedto the baseline. The cytokine level in the supernatants of peripheral blood mononuclear cells(PBMC) after ex vivo LPS stimulation demonstrated the N2-fold decrease in TNF production 4 hafter IVIG. The TNF expression, which is higher in the CD14+CD16++ monocytes, was decreased inthese cells by IVIG in 4/7 CVID cases. In vitro exposure of the healthy individuals' monocytes tothe IVIG preparation resulted in reduced TNF production, which was overcome by blockade of theFcγRIIB in the CD14+CD16++ CD32Bhigh monocytes. Our data suggest that reduction in the numberof CD14+CD16++ monocytes and the blockade of their cytokine production via triggering CD32Bcan contribute to the anti-inflammatory action of IVIG.© 2011 Elsevier Inc. All rights reserved.

⁎ Corresponding author.E-mail address: misiedla@cyf-kr.edu.pl (M. Siedlar).

1521-6616/$ – see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.clim.2011.01.002

ava i l ab l e a t www.sc i enced i r ec t . com

C l i n i ca l Immuno logy

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Clinical Immunology (2011) 139, 122–132

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1. Introduction

The cells of the monocyte lineage derive from myelomonocyticstem cells in bone marrow. They mature to monocytes, enterblood, circulate and then migrate into the tissues. In thedifferent tissues these cells (now termed macrophages)differentiate into phenotypically and functionally distinct celltypes, like alveolar and peritoneal macrophages, osteoclasts,microglia cells, etc. While the heterogeneity of tissue macro-phages is well established, monocyte heterogeneity, beforeintroduction of monoclonal antibodies (mAbs) and flow cyto-metry, has been initially defined by the presence or absence ofthe high affinity Fc-receptor [1], which later was classified asCD64. Using mAbs against the CD14 and CD16 (Fc gamma IIIAreceptor [FcγRIIIA]) molecules, human monocytes were thensubdivided into two subpopulations: CD14++CD16­ (classical) andCD14+CD16++ (non-classical or “proinflammatory”) cells [2]. TheCD14+CD16++ subset represents approximately 10% of totalblood monocytes, i.e. about 50 cells/μl. In comparison to themain subpopulation of classical monocytes, this subset ischaracterized by an enhanced expression of human leukocyteantigens (HLA) class II, CD11a, CD11c, CD18, Toll-like receptors(TLR)-2, and increased ability to produce proinflammatorycytokines (tumor necrosis factor [TNF], interleukin [IL]-12),whereas classical monocytes produce higher amounts of IL-10andmoderate levels of TNF [3–7]. The CD14+CD16++ monocytesmaybe also identified in anotherway as CD14+HLA-DR++ cells [5]and they develop into tissue macrophages or preferentially intodendritic cells [8,9].

The numbers of CD14+CD16++ monocytes are dramaticallyincreased in patients with sepsis, but are also elevated inHIV-1 infection, asthma, sarcoidosis, peridontitis, atopiceczema, pancreatitis, atherosclerosis, hemodialysis, alveo-lar proteinosis, rheumatoid arthritis, hemophagocytic lym-phohistiocytosis and Kawasaki disease [3,10]. On the otherhand, CD14+CD16++ monocytes are depleted by treatmentwith glucocorticoids [11,12]. These findings suggest that thenon-classical CD14+CD16++ monocytes are of great impor-tance in inflammatory diseases being the main source of TNFamong leukocytes, and that they may be an important targetfor anti-inflammatory therapy.

Someof the inflammatory diseases that arecharacterizedbyan increased number of CD14+CD16++ monocytes, like hemo-phagocytic syndrome [13,14] and Kawasaki disease [15,16], aretreated with high doses of intravenous immunoglobulins (IVIG)[17,18]. We hypothesized that IVIG treatment may act on theCD14+CD16++monocytes. Therefore, thepresent study on the invivo and in vitro effects of IVIG on numbers and function ofmonocyte subsets was undertaken. The CVID patients wereinvestigated, as they receive regular immunoglobulin substitu-tion. Here we show that IVIG at even supplementary doses canstrongly reduce numbers of the non-classical CD14+CD16++

monocytes and also their proinflammatory cytokine productionvia triggering of FcγRIIB (CD32B).

2. Materials and methods

2.1. Patients

We studied adult (n=18, average age – 36.2 ±13.7 years,average age at diagnosis – 18.2±19.8 years, M/F=8/10)

patients with common variable deficiency (CVID) [19] and,as a control, 17 age-matched healthy adults (average age –34.1 ±6.7 years, M/F=10/7). All patients were undersupervision of the outpatient units of the Department ofClinical Immunology and the Department of Internal Medi-cine and Gerontology of the Jagiellonian University MedicalCollege. During the study period they presented noclinical signs of acute or chronic infection (no elevated CRPand sedimentation rate) and received no antibiotics or anyadditional medication. Flow cytometry demonstrated pres-ence of CD19+ B cells in all patients studied (188 ±127 cells/μl). Therewas no evidence of bronchiectasis, splenomegaly andautoimmunity in the patients. One patient had granulomatousdisease and the number of CD14+CD16++ monocytes was in thenormal range. All patients were treated with Flebogamma IV®

(Instituto Grifols S.A., Barcelona, Spain) IVIG preparations indoses of 400 mg/kg b.w., which was given by intravenousinfusion over a 1.5 to 3 h time period. Peripheral blood wasdrawn before and after the infusion (after 4 h and in somepatients after 20 h) into polypropylene tubes containingethylenediaminetetraacetic acid– EDTA or heparin (VacutainerSystem®; Becton Dickinson Biosciences, San Jose, CA [BD]).Informed consent was obtained from patients. The study wasapproved by the Bioethical Committee of the JagiellonianUniversity Medical College (decision no. KBET/107/B/2006).

2.2. Monocyte staining and analysis

Blood samples (200 μl) werewashed by addition of 3 ml of 0.9%NaCl in polypropylene round-bottom tubes (BD) and centri-fuged (1000 × g). Then, 100 μl of the cell suspension was putinto a TruCOUNTTM Tube (BD) and stained on ice for 30 minwith 5 μl of: anti-CD45-APC (clone 2D1, #340910; BD), anti-HLA-DR-PerCP (clone L243, #347402; BD), anti-CD14-FITC(clone MY4, #6603511; Beckman-Coulter, Brea, CA), andanti-CD16-PE (clone B73.1/Leu11c , #332779; BD) mAbs. Thesamples were then treated with 400 μl of FACS Lysing Solution(BD) for 3–5 min until erythrocytes were lysed and the cellswere immediately processed in the FACSCanto flow cytometer(Immunocytometry Systems, BD) along with 10,000 of beadsper tube, and then analyzed with FACSDiva Software (BD).The absolute numbers of CD14+CD16++/CD14+HLA-DR++ andCD14++CD16­ monocytes were calculatedwith reference to thebead count. For analysis of FcγRIIB surface expression onmonocyte subpopulations anti-CD32B-AlexaFluor 488mAb wasused – a chimeric immunoglobulin withmouse IgG1(κ) variablepart and human IgG1 Fc domain, with the mutation thateliminates binding of the Fc-portion to FcγR (10 μg/ml, clone2B6, kindly provided by MacroGenics Inc., Rockville, MD) [20].This antibody was used in combination with: anti-HLA-DR-PerCP (BD), anti-CD14-APC (clone M5E2, #555399, BD), andanti-CD16-PE-Cy7 (clone 3G8, #557744, BD). An isotype controlfor anti-CD32B mAbs, AlexaFluor 488-conjugated (10 μg/ml,clone N297Q, chimeric IgG1; MacroGenics Inc., admixed withthe anti-CD14, -CD16 and -HLA-DR antibodies), was run inparallel. Electronically controlled pipettes (Eppendorf,Hamburg, Germany) were used in these procedures. Theresults for particular monocyte subpopulations are shown asdelta median of fluorescence intensity (ΔMFI) defined as a MFIof the isotype control staining subtracted from a MFI of anti-CD32B-stained cells.

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2.3. Measurement of cytokines production

Blood samples were collected in EDTA containing tubes (BD)and peripheral blood mononuclear cells (PBMC) were isolatedon a standard Ficoll/Isopaque (Pharmacia, Uppsala, Sweden)density gradient centrifugation. Isolated PBMC (1 × 105/well)were cultured with or without ultrapure lipopolysaccharide(LPS) (100 ng/ml, from Salmonella abortus eqiu S-form,#581-009-L002, Alexis Biochemicals, Lausen, Switzerland) orphytohemagglutinin (PHA) (2.5 μg/ml, Murex Diagnostics, Dart-ford, UK) for 18 h (37 °C, 5% CO2) in flat-bottom 96-microtiterplates (Nunc, Roskilde, Denmark) in the total volume of 100 μlRPMI 1640 medium (Biochrom, Berlin, Germany) supplementedwith 2 mM L-glutamine, 200 U/ml penicillin, 200 μg/ml strep-tomycin, 1–2X nonessential amino acids (all Invitrogen LifeTechnologies, Gaithersburg, MD), OPI supplement (containsoxalacetic acid, sodium pyruvate, and insulin, Sigma-Aldrich,Munich, Germany) and low-LPS fetal bovine serum (FBS) (10%,#A15-152, PAA, Pasching, Austria). Concentrations of cytokines(TNF, IL-12p70 and IL-10) in the culture supernatants or inplasma of patients were analyzed using Cytokine Bead Array(CBA) method and the flow cytometer (FACSCanto). Thefollowing Flex Sets (BD) were used: TNF (#558273), IL12p70(#558283) and IL10 (#558274). The specified beads werediscriminated in FL-4 and FL-5 channel while the concentrationof individual cytokine was determined by the intensity of FL-2fluorescence. The amount of cytokines was computed by usingthe respective standard reference curve and FCAP Arraysoftware (#641488, BD Biosciences).

2.4. Determination of intracellular TNF expression

Intracellular TNF expression was analyzed by flow cytome-try. The whole blood samples were collected into polypro-pylene tubes containing Heparin (BD) and cultured (200 μl) invitro alone or with ultrapure LPS (100 ng/ml) in the presenceof Brefeldin A (10 μg/ml, Sigma) for 4 h (37 °C, 5% CO2) inpolystyrene tubes (BD). After stimulation, erythrocytes werelysed with ammonium chloride buffer and the cells werewashed and resuspended in 100 μl of PBS. For immunostain-ing of the cell surface determinants, the following mAbswere used: anti-HLA-DR-PerCP (BD), anti-CD14-FITC (Beck-man-Coulter) and anti-CD16-APC-Cy7 (clone 3G8; #557758,BD). After incubation with mAbs (5 μl of each mAb, 20 min,4 °C) and washing in PBS, the cells were fixed/permeabilizedwith Cytofix/Cytoperm (BD) reagent (20 min, 4 °C), washedtwice with Perm/Wash solution (BD) and stained (30 min,4 °C) with anti-TNF-PE mAbs (0.5 μg/50 μl of cell suspension,clone MAbs11, #554513, BD). A PE-conjugated isotypecontrol (IgG1, BD) was run in parallel. Next, the cells werewashed twice in PBS with 0.1% bovine serum albumin (Sigma)and resuspended in PBS for flow cytometry analysis. Theresults for particular monocyte subpopulations are shown asΔMFI defined as a MFI of unstimulated cells subtracted from aMFI of stimulated cells. In some experiments, blood samplesof healthy volunteers were incubated for 30 min in thepresence of Flebogamma IV® preparation, at 30 mg/ml finalconcentration, followed by addition of LPS at 100 ng/ml andthen cultured for 4 h at 37 °C in the presence of Brefeldin A.Cells were then stained for cell surface markers andintracellular TNF, as above. For blockade of FcγRIIB, blood

samples were first incubated for 20 min at 4 °C with theunconjugated 2B6 mAb at 5 and 10 μg/ml or with the mouseisotype control (IgG1, BD) at a concentration of 10 μg/ml.This was followed by addition of LPS, culture for 4 h at 37 °Cin the presence of Brefeldin A and analysis of TNF expressionby intracellular staining and flow cytometry. In theseexperiments, the results for particular monocyte subpopula-tions are shown as ΔMFI defined as a MFI of IgG1-PE isotypecontrol stained cells subtracted from a MFI of anti-TNF-PEstained cells. The Endosafe Limulus Gel Clot assay (CharlesRiver Breeding Laboratories, Sulzfeld, Germany; sensitivity:0.06 EU/ml) was used for monitoring of LPS contamination inall media used for culture and washing.

2.5. Statistical analysis

Statistical analysis was performed by nonparametric Mann–Whitney test in all experiments using GraphPad InStatSoftware (GraphPad Soft Inc., La Jolla, CA). The resultswere expressed as mean±SD. Differences were consideredsignificant at p b0.05.

3. Results

3.1. Identification of monocyte subpopulations

Monocyte subpopulations were determined in a 4-color flowcytometry single platform assay using anti-CD45, -HLA-DR,-CD14, -CD16 mAbs and TruCOUNTTM tubes for determinationof the absolute number ofmonocytes. As it is shown in Fig. 1A,a threshold was set on CD45 staining in order to exclude non-leukocyte events and in the CD45/side scatter plot a gate(G1) was set around monocytes extending into the upperregion of the lymphocyte population. Events in this gate wereshown in a CD14/HLA-DR plot and here a gate (G2) wasdefined including CD14-positive and HLA-DR-positive events(Fig. 1B). G2 is set such that it excludes the CD16-positiveHLA-DR-negative NK cells. For G2 the pattern of CD14 andCD16 staining is shown in Fig. 1C. Here, the two monocytesubsets, i.e. the classical CD14++CD16­ monocytes and thenon-classical CD14+CD16++ monocytes are defined and theirabsolute count is determined with reference to the countingbeads shown in Fig. 1A. In the example shown there are 262CD14++CD16­ monocytes and 36 CD14+CD16++ monocytesper μl. As another way of approximate determination ofCD14+CD16++ monocytes [5], a gatewas set on CD14+HLA-DR++

cells (G3 in Fig. 1B) and this gave a value of 37 monocytes perμl in this example. In average of 18 cases of adultCVID patients, the absolute number of CD14+CD16++ andCD14++CD16­ monocytes was no significantly different fromthe number found in healthy controls (66 ± 39 and 417 ± 242versus 64 ± 31 and 549 ± 233 monocytes per μl, respectively).

3.2. Effect of IVIG infusion on monocytesubpopulations

We then studied monocyte subpopulations before and afterIVIG infusion in 18 adult patients with CVID. As shown in theexample (Fig. 2), there was a decrease of the CD14+CD16++

monocytes from 36/μl before to 10/μl at 4 h after the

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infusion. In order to exclude the possibility that the decreasewas due to occupancy or down-regulation of the CD16molecule, we analyzed also CD14+HLA-DR++ cells (Fig. 2C and

D). The number of non-classical monocytes defined in thisway also decreased from 37 to 17/μl. At the same time, theabsolute number of CD14++CD16­ monocytes remained

Figure 1 Determination of the absolute number of monocytes with the one platform assay. The cells were stained in TruCOUNT™Tubes with anti-CD45-APC, -CD14-FITC, -CD16-PE, and -HLA-DR-PerCP mAbs. The CD45-positive monocytes (G1 – “monocytic gate”)together with adjacent lymphocytes, including NK cells, were further analyzed (plot A). These cells were then gated to excludeCD14­HLA-DR­ NK cells (plot B – cells outside G2) and finally analyzed for CD14 and CD16 expressions (plot C). The absolute numbers ofCD14+CD16++, CD14+HLA-DR++ and CD14++CD16­ monocytes were estimated based on acquired bead count (10.000 in each sample). TheCD14+HLA-DR++ monocytes (brown events in plot B – G3) and CD14+CD16++ monocytes (pink events in plot C) were considered torepresent the same cell population. The calculated numbers of CD14+HLA-DR++ monocytes (plot B) were lower in some patients, incomparison to CD14+CD16++ monocytes (plot C), because a small portion of the CD14+CD16++ monocytes extends into the CD14­HLA-DR++

B lymphocytes and a few cells overlap with the CD14++ monocytes (compare plot C and B) and therefore are outside the G3 gate.

Figure 2 IVIG diminish the number of CD14+CD16+/CD14+HLA-DR++ monocytes. The significant (over 3-fold) decrease of CD14+CD16++

(A vs. B) and CD14+HLA-DR++ (C vs.D)monocytes was observed after IVIG infusion,whereas the number of CD14++CD16­monocytes (A vs. B)was unchanged. One representative data set, out of 18 performed, with cells obtained from CVID patient is presented.

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unchanged. The average decrease of CD14+CD16++ mono-cytes in 18 patients after infusion of IVIG was from 70 ±37before to 28 ±23 cells/μl (p b 0.05, Fig. 3A). The CD14+HLA-DR++ cells showed a similar pattern (Fig. 3B), while the CD14++CD16­ monocyte number remained unchanged (437± 231 and368± 266, respectively) (Fig. 3C). In order to define thekinetics of CD14+CD16++ monocyte depletion after IVIG, westudied 4 cases at 0, 4 and 20 h. The absolute count of thesecells decreased again at 4 h, but at 20 h returned towards thepre-infusion level, whereas CD14++CD16­ monocytesremained unchanged in most cases (Fig. 3D and E). Repeatedanalysis has been additionally performed on 8 patients fromthis group showing that the depletion of the CD14+CD16++

monocytes after IVIG is reproducible. As the decrease of theCD14+CD16++ monocytes may eventually be connected withthe down-regulation or shedding of the CD14 molecule fromthese cells, we checked the dot plots to see whether therewas an increase in the number of CD16-only (CD14­CD16++)cells in the monocytic gate. The average number of thissubpopulation of cells for 18 patients was 89 ±52 cells/μlbefore and 38 ±25 cells/μl after IVIG. Hence, there is noevidence for such a mechanism. Moreover, the number of NKcells that form CD14­CD16++ cell subpopulation is alsodecreased after IVIG (data not shown).

3.3. The effect of IVIG infusion on cytokine production

For the study of cytokine production before and after IVIGinfusion, PBMC of 7 CVID patients were stimulated with LPSand culture supernatants were analyzed for TNF, IL-12p70

and IL-10 content. Both TNF and IL-12p70 production wassignificantly reduced approximately 2-fold (Fig. 4A and B)after IVIG, while IL-10 production was unchanged (Fig. 4C).Similar data were obtained after stimulation of PBMC withPHA (data not shown). These findings suggest that IVIGmainly acts on the CD14+CD16++ monocytes, since these cellsare the major producers of the proinflammatory cytokines,while they produce little or no IL-10. In additional experi-ments, we analyzed LPS-stimulated cytokine production byPBMC obtained from 4 randomly chosen patients before, 4 hand also 20 h after IVIG. In 20th hour levels of IL-12 and IL-10were comparable with these before IVIG. However, produc-tion of TNF was even lower, comparing to that in 4th hourafter IVIG, and also (pb0.05) to that before IVIG (before –944.88 ± 448,41 pg/ml; 4 h after – 453.71 ± 340.27 pg/ml;20 h after – 240.23 ± 46.83 pg/ml; cytokine production bynon-stimulated PBMC was ranging b10 pg/ml). This sug-gested another (except down-regulation of the number ofnon-classical monocytes), more prolonged anti-inflammato-ry mechanism governing TNF production after IVIG. We havealso measured TNF, IL-12p70 and IL-10 in plasma samples ofthese patients before and 4 h after IVIG. Their level was notelevated in both situations (all ranged b10 pg/ml; lowerlimit of detection – 0.7 pg/ml).

In a separate group of CVID cases the in vivo effect of IVIGon cytokine production by monocyte subsets was studiedusing intracellular staining for TNF and flow cytometryanalysis. In this purpose the blood samples of CVID patientsobtained before and after IVIG infusion were stained forintracellular TNF production. As noted before, the absolutenumber of the CD14+CD16++ monocytes was reduced by IVIG

Figure 3 IVIG diminish the number of CD14+CD16++/CD14+HLA-DR++ but not CD14++CD16­ monocytes, which returns to the basal levelat 20 h after IVIG infusion in CVID patients. The subpopulations of monocytes before and after IVIG infusion were analyzed in patientswith CVID (n=18). The mean numbers of CD14+CD16+ (A) and CD14+DR++ (B) monocytes were decreased by about 50% at 4 h after IVIG,whereas CD14++CD16­ monocytes remained unchanged (C). (D and E) The numbers of CD14+CD16++ and CD14++CD16­ monocytes weremonitored just before, at 4 and 20 h after IVIG infusion in four CVID patients who agreed to take part in the study. The level of non-classical (D) monocytes returned to the basic level at approximately 20 h after IVIG infusion, whereas level of classical monocytes(E) was unchanged in most patients.

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infusion (Fig. 5A vs. D). Also the higher production of TNF byCD14+CD16++ compared to CD14++CD16­ monocytes wasconfirmed (Fig. 5C vs. B). When looking at the effect ofIVIG on TNF production there was a substantial reduction in

this cytokine by CD14+CD16++ monocytes, with a decrease ofthe ΔMFI from 364 to 34 in this example (Fig. 5C vs. F) whilethe respective values for the CD14++CD16­ monocytes were73 and 45 (Fig. 5B vs. E). This decrease of TNF production by

Figure 4 Decrease of TNF and IL-12p70, but not IL-10, production by PBMC of CVID patients after IVIG. PBMC of 7 randomly chosenCVID patients were isolated before (­) and 4 h after (+) IVIG, stimulated with LPS (100 ng/ml) and cultured for 18 h. The concentrationof cytokines was measured in the culture supernatants. Release of TNF (A) and IL-12p70 (B) was significantly decreased after IVIGinfusion, whereas IL-10 (C) was unchanged.

Figure 5 The intracellular TNF expression decreases strongly after IVIG in LPS-stimulated CD14+CD16++ monocytes. To establish theintracellular TNF expression in monocyte subsets, the whole blood cells of CVID patients were stimulated ex vivo for 4 h with LPS(100 ng/ml) before and 4 h after IVIG, in the presence of Brefeldin A (10 μg/ml). Erythrocytes were than lysed and cells were stainedwith anti-HLA-DR-PerCP, anti -CD14-FITC, anti -CD16-APC-Cy7 mAbs and anti-TNF-PE mAbs after permeabilization. After gating ofmonocytes on the scatter dot plot, only HLA-DR-positive monocytes together with adjacent lymphocytes were further analyzed forCD14 and CD16 expression (see Fig. 1). Next, in the gates for CD14++CD16­ and CD14+CD16++ monocytes (P1 and P2, respectively)intracellular expression of TNF was analyzed. The marker on dot plots was set on the control cells which do not express intracellularTNF (not shown). The ΔMFI for TNF strongly decreased after IVIG in CD14+CD16++ (C vs. F) and only moderately in CD14++CD16­

monocytes (B vs. E). One representative experiment of 7 performed (see Fig. 6) is presented, in which pronounced decrease ofTNF-ΔMFI in CD14+CD16++ monocytes was observed.

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CD14+CD16++ monocytes was seen in 4 out of 7 cases, whileCD14++CD16­ monocytes showed a moderate TNF reductiononly in 1 case (Fig. 6).

3.4. Role of CD32B in IVIG-induced suppression ofTNF production by monocyte subsets

One of the potential mechanisms of the anti-inflammatoryaction of IVIG is via binding to Fcγ receptors. While most of Fcreceptors trigger signaling cascades that lead to activation ofleukocytes, the FcγRIIB (CD32B) can suppress cell activity,due to stimulation of ITIM motifs in its cytoplasmic domain[21]. Therefore, the expression of this receptor on monocytesubpopulations of healthy donors was analyzed. While abouthalf of the CD14++CD16­ monocytes showed expression ofCD32B, almost all CD14+CD16++ monocytes were positive(Fig. 7B and C, respectively). When comparing ΔMFI of thesetwo subsets, the average expression of CD32B in 4 samplesanalyzed was 3-fold higher on CD14+CD16++ monocytes(Fig. 7G). After IVIG infusion no change in the expression ofCD32B was observed in the non-classical CD14+CD16++

monocytes, although their number was substantially reduced(Fig. 7C vs. F). For the classical CD14++CD16­ monocytes thelevel of CD32B expression in this particular example wasdown regulated (Fig. 7B vs. E), but in average of 4experiments there was no statistically significant decrease(data not shown). The expression level of CD32B variedbetween individuals, but the difference in expressionbetween the two monocyte subsets was very pronounced inevery case.

We then asked, whether blockade of CD32B would be ableto overcome the inhibitory effect of IVIG on TNF expression byCD14+CD16++ monocytes. In this purposewe performed a set ofin vitro experiments on peripheral blood of healthy donors(n=7). The blood samples were incubated in vitro with IVIGpreparation for 30 min (followed by LPS stimulation) then TNFproduction, as assessed by intracellular staining, was reduced

about 4-fold in CD14+CD16++ monocytes (Fig. 8A). Pre-incubation for 20 min with unconjugated anti-CD32B mAbs at5 μg/ml led to a partial and at 10 μg/ml to complete recoveryof TNF production. Analysis of CD14++CD16­ monocytes in thesame samples showed a moderate reduction of TNF expres-sion, but this was not overcome by anti-CD32B mAbs (Fig. 8B).These data suggest that among monocytes, the CD14+CD16++

subset is the major target of IVIG-induced suppression of TNFproduction, an effect mediated by CD32B.

To ascertain that IVIG do not inactivate LPS activity, the10 and 100 ng/ml of LPS were (in 3 experiments performed)mixed with IVIG preparation at 30 mg/ml. Immunoglobulinsdid not affect the coagulation of the gel in the Limulus assay.Also LPS-induced (at 100 ng/ml) TNF production by the cellsof monocytic MonoMac 6 line was unaffected by addition ofIVIG at the 30 mg/ml dose (data not shown).

4. Discussion

In this study a new labeling approach to enumerateCD14++CD16­ and CD14+CD16++ monocyte subsets in the bloodwas used [22]. Apart from the adequate identification of thesesubpopulations this procedure allows for efficient exclusion ofNK cells from analysis. The 4-color flow cytometry and a singleplatform assay were used to determine the absolute numbersofmonocyte subsets in peripheral blood.Anadditionalwashingstep before staining of the whole blood samples wasintroduced, because in some patients, especially after IVIGinfusion, the fluorescent intensity of CD16 staining was weak,resulting in difficulties in defining CD14+CD16++ monocytesproperly. This may be due to the fact that binding sites ofanti-CD16 mAbs of IgG1 isotype (Leu11c/B73.1, 3G8, andBL-LGL/1 clones), and also F(ab’)2 fragments of 3G8 mAbs onthe CD16 (FcγRIIIA) molecule overlap with the binding sites ofeither monomeric or polymeric IgG, as has been shown in caseof CD16high NK cells [23]. Therefore, washing step used hereinappears to be crucial for preventing interference of IgG inserum and/or IVIG preparations with binding of anti-CD16mAbs.

During inflammation the number of CD14+CD16++ mono-cytes increases [3]. Since patients with humoral immunodefi-ciencies often suffer from various bacterial infections, welooked first at the numbers of classical and non-classicalmonocytes in 18 CVID patients and showed that the numbers ofmonocytes of both subpopulations did not significantly differfrom 17 control subjects. These findings do not exclude thepossibility that monocyte subsets may be altered with severeinfections in CVID. Since there is evidence for strongermacrophage activation in nodular intestinal lymphoid hyper-plasia observed in some CVID patients [24], we especiallylooked at the 1 patient with this type of clinical presentation,but again the number of CD14+CD16++ monocytes was withinthe normal range (84/μl). Another two patients with highnumbers of non-classical monocytes presented an increase ofall monocytes, i.e. their CD14++CD16­ monocytes wereincreased as well. The origin of this monocytosis is unknown.

When we analyzed the numbers of monocyte subpopula-tions after IVIG infusion in CVID patients, we found a decreaseonly in the CD14+CD16++ subpopulation, which was about 50%at 4 h after IVIG, whereas CD14++CD16­ monocytes remainedunchanged or decreased moderately in a few patients.

Figure 6 Intracellular expression of TNF in LPS-stimulatedCD14+CD16++ monocytes decreases after IVIG only in some CVIDpatients. In 7 CVID patients expression of intracellular TNF wasanalyzed ex vivo. ΔMFI was measured before and 4 h after IVIG inboth subpopulations of monocytes. ΔMFI of CD14+CD16++ mono-cytes decreased in 4 patients and in CD14++CD16­ only slightly in1 patient.

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The mechanisms responsible for reduction of the numberof CD14+CD16++ monocytes are unclear. One plausiblemechanism of IVIG-induced depletion of CD14+CD16++ mono-cytes from periphery is by apoptosis. It was suggested thatanti-Fas antibody-mediated Fas (CD95/APO-1) ligation andactivation of caspases may be involved in this process, sincein IVIG preparations both, agonistic and antagonistic anti-CD95 antibodies are present in low titers that are quickly“consumed” after IVIG infusion. Induction of apoptosis byIVIG may be caused also by the concerted action of a range ofother, poorly defined, different antibodies [25–27]. Usingproteomic and transcriptomic methods Zhao et al. providedevidence that pro-apoptotic genes are constitutively up-regulated in CD16+ monocytes, which might make these cellsparticularly susceptible for apoptotic death [28]. The IVIG-

induced apoptosis of CD14+CD16++ monocytes might be alsodue to ligation and cross-linking of CD32B. A CD32B-mediatedapoptosis in B lymphocytes has been reported [29] and a rolefor CD32B has been shown for IgG-induced apoptosis ofplasma cells [30].

It has been noticed that the numbers of CD14+CD16++

monocytes returned to the basic level in ca. 20 h after IVIG.The CD14+CD16++ cells need for their maturation macro-phage - colony stimulating factor (M-CSF) which is stablypresent in circulation [31] and the half life of monocytes inthe blood stream is of approximately 24–48 h [32]. Thereforeit is plausible that CD14+CD16++ monocytes rapidly downregulated after IVIG infusion need this time (ca. 20 h) to bereplenished by the classical monocytes maturing in thepresence of M-CSF.

Figure 7 CD32B is highly expressed on CD14+CD16++ monocytes. For determination of surface expression of FcγRIIB (CD32B) onmonocyte subpopulations, the whole blood samples of CVID patients were stained ex vivo (before and 4 h after IVIG) with anti-FcγRIIB-AlexaFluor488, anti-HLA-DR-PerCP, anti-CD14-APC and anti-CD16-PE-Cy7 mAbs. Appropriate AlexaFluor488 conjugated isotypecontrol was run in parallel. Monocytes were gated on scatter dot plot and the both subpopulations were next defined as shown on theFig. 1. The marker on dot plots was set on the cells stained with isotype control (not shown). The percentage of CD32B-positive cellsand ΔMFI for CD14++CD16­ (B and E) and CD14+CD16++ (C and F) monocytes are shown. One representative staining of four performedbefore (A–C) and 4 h after IVIG (D–F) is shown. The ΔMFI of anti-CD32B mAbs-stained cells (G) was approximately 3 times higher fornon-classical monocytes, in comparison to classical ones, referring to the cells before IVIG infusion (n=4, ±SD).

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In present study, in patients receiving IVIG doses of400 mg/kg b.w., a pronounced decrease in the absolutenumber of CD14+CD16++ monocytes was observed. Foranti-inflammatory treatment, 5-times higher total dose ofIVIG preparations for 5 consecutive days is used, forinstance in Kawasaki syndrome [18]. Analysis of monocytesubpopulations using this high dose (combined IVIG withaspirin and pentoxifylline) has shown an average 5-folddecrease in initially elevated non-classical monocytesthat maintain in the normal range until day 30 after IVIG[33].

The use of IVIG at 400 mg/kg b. w., as immunoglobulinreplacement therapy, caused reduction of TNF and IL-12p70production of LPS- or PHA-stimulated patients’ PBMC,whereas IL-10 level remained unchanged. As CD14+CD16++

monocytes are the main source of TNF in man [5], and sincethey produce little IL-10 [34], we hypothesized that a mainanti-inflammatory action of IVIG might be targeting ofCD14+CD16++ monocytes. In fact, after IVIG infusion apronounced decrease of TNF production in ex vivo LPS-stimulated CD14+CD16++, but not in CD14++CD16­ monocyteswas observed. The effect of IVIG on TNF production byCD14+CD16++ monocytes, as assessed by intracellular stain-ing, was not seen in all cases. On the other hand, TNF releaseby LPS- or PHA-stimulated PBMC of CVID patients wasconsistently reduced in all subjects. This difference may bebest explained by the depletion of the CD14+CD16++

monocytes, as the main producers of TNF. Hence, thereduced TNF production in PBMC supernatant after IVIGtreatment can be explained by a) a depletion of the TNF-producing CD14+CD16++ monocytes and b) in some cases byan additional reduction of the TNF production by theCD14+CD16++ monocytes remaining in blood after IVIG. Inthe other patients, TNF production by the CD14+CD16++

monocytes was refractory to IVIG. It remains to be analyzedwhether a higher dose of IVIG at 2 g/kg b.w., as used for anti-

inflammatory effect, will reduce TNF production in theseCD14+CD16++ monocytes, as well.

We then studied themechanism of this preferential actionof IVIG on CD14+CD16++ monocytes and asked whichreceptors might be involved. An inhibitory effect of pooledhuman immunoglobulins on proinflammatory cytokine pro-duction by PBMC has been reported earlier and evidence wasprovided for the inhibition of NF-κB activation [35–37]. It hasbeen suggested that the inhibitory FcγRIIB (CD32B) isinvolved in the anti-inflammatory action of IVIG [38]. Inmurine macrophages TNF production can be blocked byimmune complexes and this blockade is absent in CD32Bknock-out mice [39]. Therefore, we first analyzed theFcγRIIB expression on both subpopulations of monocytes. Infact, this receptor shows an approximately 3-fold higherexpression on CD14+CD16++, when compared to CD14++CD16­

cells. Next, the functional importance of CD32B expressionon monocyte subsets in IVIG-mediated suppression of TNFwas assessed using an anti-CD32B antibody, which is able toblock binding of IgG to this receptor [40]. Although classicalCD14++CD16­ monocytes also express CD32B, this antibodydid block the IVIG-mediated suppression of TNF in the non-classical CD14+CD16++ CD32Bhigh monocytes selectively(Fig. 8). In our in vitro experiments, IVIG doses of 30 mg/mlwere used, which is the highest level that can be reachedfollowing IVIG infusion at 400 mg/kg b.w. [25]. Albeit notsignificant, we also noted a TNF decrease at a lower dose ofIVIG (10 mg/ml) which is a level routinely observed duringsubstitution therapy (data not shown) [41].

It is known that DC-SIGN molecule on human dendriticcells is the receptor engaged in the mechanisms of anti-inflammatory activity of sialylated Fc fragments of IgG in IVIGpreparations [41,42]. However, DC-SIGN was not detectedon both subsets of monocytes (data not shown). Therefore,DC-SIGN appears to be not involved in the action of IVIG onmonocytes.

Figure 8 The inhibitory effect of IVIG on LPS-stimulated TNF synthesis in CD14+CD16++ cells is reversed, in a dose dependentmanner, by anti-FcγRIIB mAbs. To detect intracellular TNF expression in CD14+CD16++ (A) and CD14++CD16­ (B) monocytes, appropriateblood samples were incubated in vitro for 20 min with unconjugated anti-FcγRIIB mAbs (5 and 10 μg/ml) or mouse IgG1 isotype control(10 μg/ml), then for 30 min with IVIG (30 mg/ml) and finally 4 h with LPS (100 ng/ml). The cells were labeled with anti-HLA-DR-PerCP,anti-CD14-FITC, anti-CD16-APC-Cy7 mAbs and, after permeabilization, with anti-TNF-PE mAbs or isotype control (IgG1-PE). The resultsare presented as a mean values of ΔMFI (n=7, ±SD). In 4 additional control experiments IVIG and/or anti-CD32B mAbs were added tothe whole blood and cultured for 4h; in such approach, there was no TNF production by monocyte subpopulations observed (data notshown).

130 M. Siedlar et al.

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In conclusion, we show that IVIG substitution selectivelyreduces the number of non-classical CD14+CD16++ mono-cytes. Furthermore, it can block TNF production by thissubset by a mechanism involving CD32B. Hence, our datashow that among monocytes, the CD14+CD16++ cells are themajor target for the transient (at least within 20 h) anti-inflammatory activity of IVIG. These data also suggest thatthe CD14+CD16++ monocytes may be involved in the anti-inflammatory action of IVIG in conditions like Kawasakidisease [33,43].

Acknowledgments

We wish to thank Ms. Mariola Ożóg for skillful technicalassistance.We acknowledgewith thanks the gift of anti-CD32BmAbs by MacroGenics Inc. This work was supported by theNational Committee for Scientific Research grant No. 3220/PO1/2007/32. M. Siedlar was supported by the Alexander vonHumboldt Foundation. L. Ziegler-Heitbrock was supported bygrant Zi288 from Deutsche Forschungsgemeinschaft.

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Clinical Immunology (2011) 139, 105–106

EDITORIAL

Another new mechanism of action of IGIV☆

Since the initial report by Paul Imbach in 1981 that highdoses of IGIV increased the platelet counts of patients withidiopathic thrombocytopenic purpura [1], and the report ofdramatic effects of high doses of IGIV in Kawasaki syndrome[2], the use of pooled polyclonal human IgG preparations hasincreased continuously as this mode of therapy has beenapplied to an ever-broadening spectrum of auto-immune andinflammatory diseases [3]. In parallel with this increasedusage of IGIV for conditions that affect diverse organsystems, there have been continually increasing effortstowards understanding “the” mechanism of action of IGIV[3–5]. While most physicians treating autoimmune andinflammatory diseases with IGIV still use doses in the rangeof 1–2 g/kg as reported by Imbach, there has been a steadyincrease in the doses of IgG used for replacement therapy inantibody deficiency syndromes. Few PID patients are stilltreated with the 100 mg/kg/month recommended by theBritish Medical Research Council in 1971 [6,7]. However,there have been few dose-response studies with IGIV inautoimmune/inflammatory diseases.

It has become obvious that IGIV treatment has manyeffects besides neutralizing bacterial toxins and viruses and/or promoting opsonophagocytic killing of microorganisms.However, much less is known about how immunomodulatoryeffects of IgG contribute to immunologic homeostasis innormal individuals. Similarly, the extent to which abnormal-ities in immunomodulatory networks might contribute to thepathology in primary immune deficiency diseases (PIDD) suchas common variable immune deficiency (CVID) remainsunderexplored.

In this issue, Siedlar et al. report that the numbers ofperipheral blood monocytes belonging to the CD14+CD16++subset and considered “proinflammatory” [8,9] are decreasedby IGIV treatment in vivo, and that IGIV also suppressesproduction of pro-inflammatory cytokines such as TNF inresponse to LPS in vitro [10]. A particular strength of this paperis the concordance of in vivo, ex vivo and in vitro observations:first, the authors show that the CD14+CD16++ subset ofmonocytes decrease in peripheral blood of CVID patientsafter IGIV treatment; second, they show that peripheral blood

☆ Disclosure: The author is salaried employee of CSL Behring withequity interest.

1521-6616/$ – see front matter © 2011 Elsevier Inc. All rights reserveddoi:10.1016/j.clim.2011.02.002

mononuclear cells taken from CVID patients 4 hrs after IGIVinfusions and stimulated ex vivowith LPS produce less TNF andIL-12p-70 than cells obtainedbefore an IGIV infusion; third, theyshow that IGIV treatment in vitro also decreases the TNFproduction in response to LPS by peripheral blood monocytesfrom healthy individuals. The production of IL-10 was notaffectedby the IGIV treatment, and effects onCD16-monocyteswere much less dramatic. The relationship between theobservations of decreased numbers of the pro-inflammatorysubset of monocytes in the blood of CVID patients after IGIVtreatment and decreased production of pro-inflammatorycytokines by these cells ex vivo and in vitro is not clear. Inaddition, the durability of these effects in vivo and the role ofIVIG dose on the described changes also need to be furtherdelineated. However, taken together, the results suggest thatdecreasing cytokine production by a particularly pro-inflamma-tory subset ofmonocytes is a real biological effect of IGIV.Whatis the mechanism of this effect and what is its significance?

To approach the question of the mechanism by which IGIVsuppressed the cytokine production, the authors studied theexpression and role of CD32B, a type of FcγRIIB which hasinhibitory rather than activating tyrosine motifs in itscytoplasmic tail [11,12]. The authors observed that theCD14+CD16++ subset of monocytes had 2–3 fold higherexpression of CD32B than CD16− monocytes, and went on toshow that antibody to CD32B blocked, in a dose dependentfashion, the inhibitory effects of IGIV on LPS induced TNFproduction using cells from healthy volunteers in vitro. Theparticular anti-CD32B used was a chimeric molecule with amutation in the Fc region which prevents that part of themolecule from interacting with FcγRs [13]. Induction ofCD32B expression by a sialylated subset of molecules in IGIVpreparations has been postulated to be a major mechanismof anti-inflammatory effects of IGIV [14,15]. That effect isbelieved to be mediated by DC-SIGN [16]. However, theCD32B-mediated effects in the paper of Siedelar et al. wereseen in vitro, and the authors report that they could notdetect DC-SIGN expression on their monocyte preparations.

There are numerous reports of mechanisms of action ofIGIV in autoimmune and inflammatory diseases [reviewed in3–5]. These include neutralization of pathogenic antibodiesby anti-idiotypes; neutralization of antigens, superantigensand/or bacterial toxins; inhibition of complement deposition

.

106 EDITORIAL

on targets and decreased anaphylotoxin function; formationof immune complexes which block phagocytosis, increasedcatabolism of pathologic IgG molecules, modulation of FcRfunction, inhibition of cytokine production, induction ofapoptosis, and modulation of T-cell activation, just to namea few. However, data which allow us to determine theimportance of the role played by any individual mechanismor mechanisms in the therapeutic effect of IGIV in any givendisease in humans are much more difficult to obtain. In thiscontext, several points in the paper of Siedlar et al. areworthy of comments. First of all, the effects were observedin most but not all CVID patients after they received astandard replacement dose of IGIV, 400 mg/kg. Inhibition ofTNF production in response to LPS in vitro was achieved with30 mg/ml of IGIV, a concentration which could be reachedduring “high dose” therapy at 1–2 g/kg, as used in the anti-inflammatory and autoimmune diseases, but which isunlikely to be reached or sustained in routine replacementtherapy for primary immune deficiencies. Together withobservations reported elsewhere showing increases in theCD14+CD16+ subset of monocytes in the blood of patientswith Kawasaki syndrome and other systemic inflammatoryconditions [17,18], these results suggest that the mechanismillustrated in this paper might, in fact, be important in thedramatic effect of IGIV in most patients with those condition.A more tantalizing possibility is suggested by the observationthat treatment of CVID patients with regular replacementdoses of IGIV in vivo decreased the abundance of theproinflammatory monocyte population in their blood for aslong as 20 hrs in some patients. It is well recognized that CVIDpatients are at increased risk for granulomatous disease andother chronic inflammatory conditions. Could the increasedincidence of those complications of antibody deficiencies bedue to a decrease in an anti-inflammatory effect of IgGenjoyed by their “normal” counterparts? The lower inci-dence of such complications in frankly agammaglobulinemicpatients suggests not, but there may be other reasons forthat. Conversely, could achieving or maintaining higherserum IgG concentrations decrease the incidence or severityof inflammatory complications of CVID? Could the lack ofresponse to this inhibitory effect of IGIV on cytokineproduction in some individuals with CVID be used to identifythose at highest risk for granulomatous disease and/or otherinflammatory disorders? These questions can only beanswered by studies of a broader population of wellcharacterized PIDD patients over longer periods of time.Analyses of in vivo effects whose mechanisms we canunderstand in vitro are particularly useful. However, what ismost important is the correlation of laboratory findings withclinical observations in longitudinal studies. To achieve that,increased participation in registries and accumulation of dataon well-characterized patients are the only way forward.

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Melvin BergerImmunology Research and Development, CSL Behring, LLC,

1020 First Ave, King of Prussia, PA 19406, USAE-mail address: Mel.Berger@cslbehring.com.