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. 2003 Sep;133(3):476–484. doi: 10.1046/j.1365-2249.2003.02245.x

Donor dependent, interferon-γ induced HLA-DR expression on human neutrophils in vivo

W REINISCH *, C LICHTENBERGER *, G STEGER ‡, W TILLINGER *, O SCHEINER †, A GANGL *, D MAURER §, M WILLHEIM †
PMCID: PMC1808784  PMID: 12930377

Abstract

Neutrophils are effector cells of innate immune responses. Stimulated by interferon-γ (IFN-γ) to express HLA-DR, neutrophils acquire accessory cell functions for superantigen-mediated T cell activation. In vitro HLA-DR induction on neutrophils varies in a functionally relevant way as levels of MHC class II expression and magnitude of neutrophil induced T cell responses are correlated functions. The aim of this study was to assess whether IFN-γ induces HLA-DR on human neutrophils in a donor dependent fashion in vivo and to define regulatory events operative in MHC class II expression of neutrophils. In vivo administration of rhIFN-γ in 55 patients with renal cell carcinoma resulted in a varying increase of HLA-DR on neutrophils. By setting a cut-off for response at>10% HLA-DR positive neutrophils, HLA-DR responders (51%) were as frequent as nonresponders (49%). In vivo kinetic studies revealed a peak expression of HLA-DR on neutrophils 48 h after rhIFN-γ application, while nonresponders remained HLA-DR negative over a 72-h period. In vitro IFN-γ stimulated neutrophils recapitulated the response profiles observed in vivo. No differences in IFN-γ dependent CD64 and invariant chain expression, and IFN-γ serum levels were observed among the response subgroups. HLA-DR mRNA was detected in neutrophils from rhIFN-γ treated responders and nonresponders, HLA-DR protein solely in lysates of responder neutrophils. IFN-γ stimulated HLA-DR expression on neutrophils is subject to donor dependent variations in vivo, which result from rather post-transcriptional than transcriptional regulation. Due to their abundance in inflammatory reactions heterogeneous HLA-DR expression by neutrophils could determine the outcome of superantigen-driven diseases.

Keywords: neutrophils, IFN-γ, MHC class II, CD64, invariant chain

INTRODUCTION

Major histocompatibility complex (MHC) class II antigens are cell surface glycoproteins constitutively expressed on subsets of antigen-presenting cells (APCs) including dendritic cells, monocytes, and B lymphocytes, which regulate adaptive immune responses by presentation of peptides to CD4+ T lymphocytes. In humans three MHC class II isotypes, HLA-DP, -DQ, and -DR, each composed of α- and β-chains, are coregulated on APCs in a tissue-specific and cytokine-inducible manner [1]. Cytokine-mediated induction of MHC class II molecules in inflammatory lesions confers antigen-presenting capacity also to other cell types, such as epithelial cells. This mechanism has been considered to play a pivotal role in the pathogenesis and progression of autoimmune diseases [2,3].

Interferon-γ (IFN-γ) is a major mediator orchestrating the induction of antigen processing and presentation via the MHC class II pathway [4]. By triggering a specific cell surface receptor, consisting of a high-affinity binding α-chain and a signalling β-chain, IFN-γ activates the latent cytosolic signal transducer and activator of transcription (STAT) 1α, leading to transcription of the class II transactivator (CIITA) via its promoter IV region [5,6]. As transcriptional coactivator interacting with constitutive promoter-bound transcription factors, CIITA operates as master regulator of the key genes required for cell surface expression of MHC class II antigens, namely MHC class II α- and β-chains, invariant chain (Ii; CD74), and HLA-DM [7–9]. Ii facilitates proper folding of MHC class II αβ complexes, prevents premature binding of peptides and targets the complexes to the endosomal/lysosomal pathway [10,11].

Neutrophils have long been regarded as terminally differentiated, primary effector cells restricted to innate immune reactions. However, the observation that neutrophils are capable of synthesizing MHC class II molecules and various cytokines promoted a new view on these cells as participants in adaptive immunity [12–15]. HLA-DR is inducible in human neutrophils both in vitro by IFN-γ, granulocyte-macrophage colony stimulating factor (GM-CSF), or interleukin-3 (IL-3), and in vivo by IFN-γ or GM-CSF [12,17,13,16]. During active disease neutrophils of patients with Wegener's granulomatosis have been described to acquire MHC class II antigens [18]. Data concerning the potential of neutrophils to process and present soluble antigens such as tetanus toxoid are controversial [19,20]. However, HLA-DR positive neutrophils have been shown to stimulate CD4+ T cells via superantigen which crosslinks MHC class II molecules with the variable portion of the T cell receptor α-chain. Due to the abundance of neutrophils in various inflammatory reactions the expression of HLA-DR on these cells could be essential in diseases, in which superantigens may contribute to pathogenesis such as rheumatic fever or inflammatory bowel disease [21]. Donor dependent variations in inducible HLA-DR expression on neutrophils may influence the course of these diseases as levels of neutrophil MHC class II expression and the magnitude of T cell responses are correlated functions [19].

Here we asked whether IFN-γ mediated induction of HLA-DR on human neutrophils, in vivo as well as in vitro, is subject to a donor dependent regulation and tried to define molecular mechanisms operative in the regulated expression of HLA-DR in neutrophils. Furthermore, we investigated whether inducibility of HLA-DR on neutrophils correlates with HLA-DR up-regulation on monocytes after in vivo stimulation with rhIFN-γ.

MATERIALS AND METHODS

Study design

Fifty-five patients (female/male: 27/28; median age 60 years; range 37–75 years) with histologically confirmed progressive metastatic renal cell carcinoma receiving recombinant human interferon gamma-1b (rhIFN-γ, Imukin®, Bender+Co GesmbH, Vienna, Austria, 3 × 107 U/mg) were included in the study after informed consent. The treatment schedule included cycles of rhIFN-γ administered subcutaneously at a dose of 100 µg per day three times a week for two weeks [22]. No patient had been previously treated with chemo- or immunotherapy. Immediately before (baseline) and 48 h after rhIFN-γ applications heparinized venous blood was collected for cell isolation and flow cytometric methods. White blood cell count were performed and serum collected simultaneously. For mRNA studies heparinized venous blood was drawn 24 h after rhIFN-γ administration. From six selected patients additional blood samples were obtained 4, 10, 24, 48 and 72 h after rhIFN-γ injection. Blood samples from 47 age- and sex-matched healthy donors (female/male: 21/26; median age 55 years, range 34–73) served as controls.

Antibodies

Unconjugated and FITC-labelled murine monoclonal antibody (mAb) L243 (IgG2a; Becton Dickinson, San Jose, CA) detects a conformational, nonpolymorphic HLA-DR epitope composed of α and β subunits [23]. Anti-Ii mAb VIC-Y1 recognizing the N-terminal/cytoplasmic domain of Ii was kindly provided by O. Majdic (Institute of Immunology, University Vienna, Medical School, Austria). FITC-labelled anti-FcγRI (CD64) mAb 32·2 (IgG1) was from Medarex (West Lebanon, NH, USA), anti-HLA class I mAb W6/32 (IgG2a) from Cymbus (Southhampton, UK), and R-PE-labelled anti-FcγRIIIB (CD16) mAb 3G8 from Monosan (Uden, Netherlands). FITC-labelled or unconjugated isotype-matched mAbs from Sigma (Deisenhofen, Germany) and R-PE labelled mouse IgG1κ from Pharmingen (San Diego, CA, USA) served as controls. FITC-conjugated rabbit antimouse IgG (DAKO, Glostrup, Denmark) was used for the indirect immunofluorescence staining protocol.

Measurements of peripheral blood leucocyte counts and differentials

Peripheral blood leucocyte counts and differentials were determined by a routine laboratory procedure (NE-8000 Sysmex; Toa Medical Electronics Co. LTD., Kobe, Japan).

Serum levels of IFN-γ, GM-CSF and IL-3

Serum concentrations of IFN-γ, GM-CSF, and IL-3 were measured by ELISA (Quantikine, R & D Systems, Minneapolis, MN, USA). All samples were run in duplicates. The lower detection limits were 3 pg/ml for IFN-γ, 2·8 pg/ml for GM-CSF and 7·4 pg/ml for IL-3.

Isolation and culture of peripheral blood neutrophils

15 ml of stabilizer-free heparin anticoagulated peripheral venous blood were diluted with an equal volume of PBS without Ca++/Mg++ (Life Technologies, Paisley, UK) and layered over a dual density Percoll gradient (57% v/v in PBS, d = 1·075 g/ml; 67% v/v, d = 1·088 g/ml; Pharmacia, Freiburg, Germany) modified from Venaille et al. [24] Centrifugation was performed for 60 min with 400 g at room temperature. Neutrophils were carefully collected from the 57%/67% interphase with a purity of more than 98%, washed three times with PBS, and immediately cultured at a cell density of 2 × 106/ml in RPMI 1640 (Bio Whittaker, Walkersville, MD, USA) supplemented with 10% FCS (Mycoplex; PAA Laboratories, Linz, Austria), 2 mm glutamin, 200 IU/ml penicillin and 200 µg/ml streptomycin (all Life Technologies) in the presence or absence of 0·1, 1, 10, and 100 IU/ml rhIFN-γ (Bachem Biochemica GmbH, Heidelberg, Germany; 3 × 107 IU/mg) for 24 and 44 h at 37°C in 5% CO2. In selected experiments neutrophils were stimulated with either 100 IU/ml rhIFN-γ or 200 IU/ml rhGM-CSF (Genzyme, Cambrigde, MA, USA), or a combination of both cytokines. Neutrophils were washed and exposed to human monomeric 7S-IgG (12 mg/ml, Biochemie GmbH, Vienna, Austria) to quench nonspecific binding sites.

Flow cytometry

A previously described whole-blood lysis technique was used for flow cytometric detection of surface and intracellular antigens [25]. Briefly, 90 µl of whole-blood were incubated with 10 µl of prediluted fluorochrome-labelled or unconjugated mAbs or corresponding isotype-matched controls for 30 min at 4°C. For indirect immunofluorescence staining leucocytes were preincubated with 10% rabbit immunoglobulin (DAKO), before rabbit antimouse IgG-FITC (DAKO) was added for another 30 min at 4°C. Cells were washed and resuspended in HBSS containing 0·3% bovine serum albumin (BSA; Sigma) and 0·1% sodium azide. Erythrocytes were eliminated by lysing solution (Becton Dickinson, Heidelberg, Germany). To detect cytoplasmic Ii leucocytes were fixed with 2% formaldehyde for 15 min at room temperature and permeabilized with 0·1% saponin/HBSS. Immunophenotyping of cultured neutrophils with fluorochrome-labelled antibodies was performed in HBSS supplemented with BSA and sodium azide analogous to the whole-blood lysis protocol. 15·000 cells per sample were analysed on a FACScan (Becton Dickinson) using the LYSIS II software. Unlabelled, as well as FITC- and R-PE-labelled microspheres (Becton Dickinson) were used for calibration of flow cytometer settings. Neutrophils were identified by light-scatter properties and staining with anti-CD16 R-PE [26] monocytes by their characteristic light-scatter signals only. Viability of neutrophils after culture was determined by exclusion of propidium iodide (Sigma) positive cells.

Purification of neutrophils by positive cell sorting

Neutrophils were purified according to a previously published method [26]. Additionally, neutrophils enriched by Percoll gradient were double-stained with anti-CD16 R-PE and FITC labelled anti-HLA-DR mAb L243 for 30 min at 4°C and subjected to fluorescence activated cell sorting on a FACStar Plus (Becton Dickinson). After instrument sterilization CD16bright/HLA-DR+ cells were separated to a purity exceeding 99·9% and kept on ice until morphological analysis.

Morphological cell analysis of purified neutrophils

In a total volume of 200 µl HBSS, 2 × 105 sorted CD16bright/HLA-DR+ cells were centrifuged onto slides (300 g, 3 min) using a cytocentrifuge (Shandon, Frankfurt, Germany), histochemically stained by Hemacolor (Merck, Vienna, Austria) and analysed by light microscopy on a Zeiss microscope at 1000× magnification (Göttingen, Germany).

RNA extraction and RT-PCR

Total RNA isolation from purified neutrophils, reverse transcription and PCR were performed as outlined elsewhere [26]. Primers used for PCR of HLA-DRα were as follows: sense 5′-TGGGAC CATCTTCATCATCAAGG-3′, antisense 5′-GGGCATTCCAT AGCAGAGACAG-AC-3′; as positive control β-actin was amplified using the following DNA sequences: sense 5′-GCTATCCCT GTACGCCTCTG-3′, and antisense 5′-CTCCTTCTGCATCCT GTCGG-3′.

Western blot analysis

Lysates of percoll-enriched neutrophils of patients before and 48 h after in vivo rhIFN-γ administration were studied for HLA-DR dimers. Peripheral blood mononuclear cells (PBMCs) and neutrophils of healthy donors and, neutrophils in vitro stimulated with rhIFN-γ (100 U/ml) and rhGM-CSF (100 U/ml) for 44 h served as controls. Cells were incubated in lysis buffer (2% Polidocanol, 0·1 mm phenylmethylsulphonylfluoride and 0·5 mm N-α-tosyl-l-lysine chloromethyl ketone; all from Sigma) on ice for 10 min Lysates were centrifuged for 10 min at 500 g/4°C, followed by 10 min at 16·000 g/4°C. Supernatants were diluted 1 : 2 in twofold sodium dodecyl sulphate (SDS; Sigma) loading buffer containing 4% mercaptoethanol, aliquoted, and incubated for 10 min at room temperature or 96°C before separation on discontinous 10% SDS-PAGE. Fractionated proteins from 5 × 105 cells per lane were transferred to PVDF membrane (Hybond™-P; Amersham, Uppsala, Sweden) overnight at 4°C. HLA-DR dimers were detected with mAb L243 followed by HRP-conjugated goat antimouse Ig (Amersham) both diluted with 1% BSA/PBS. Membranes were incubated with ECL Plus™ and exposed to Hyperfilm™ (Amersham).

Statistical analysis

Statistical analysis was performed using Sigmastat™ (Jandel Co., SPSS Science, Chicago, IL, USA). Continuous data are presented as median and 25th to 75th percentile (interquartile range, IQR). Δ specifies the difference between baseline and post-treatment values. Mann–Whitney Rank Sum Test was used for unpaired, Wilcoxon Signed Rank Test for paired comparison of groups. Area under the curve (AUC) was calculated by the trapezoidal rule. Association between ΔHLA-DR of neutrophils and ΔHLA-DR of monocytes was calculated by Spearman Rank Order Correlation. P-value of <0·05 was considered statistically significant.

RESULTS

Analysis of HLA-DR, CD64 and Ii expression on peripheral blood neutrophils after in vivo application of rhIFN-γ

Neutrophils were defined by their light scatter characteristics and bright immunofluorescence with anti-CD16 mAb. Neutrophils from healthy donors (n = 47) and patients at baseline (n = 42) were consistently devoid of HLA-DR surface expression. RhIFN-γ administration was followed by induction of surface HLA-DR expression in 91% (n = 50) of patients (median 10%, IQR 4–25; P < 0·001). CD16bright/HLA-DR+ cells in IFN-γ treated patients were neutrophils as determined by positive cell sorting and light microscopy (data not shown).

Heterogeneity of IFN-γ dependent HLA-DR expression on neutrophils was illustrated by setting a cut-off for HLA-DR induction at>10% positive neutrophils. Patients responding to rhIFN-γ with HLA-DR induction above the cut-off were defined as HLA-DR responders, all others as HLA-DR nonresponders. In 22/28 HLA-DR responders baseline and post-treatment expression of HLA-DR on neutrophils was measured with a median Δ%HLA-DR of 27 (IQR: 21–37), and in 20/27 nonresponders with a median Δ%HLA-DR of 4 (IQR: 0–5; Fig. 1).

Fig. 1.

Fig. 1

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Subgrouping of patients according to in vivo expression of HLA-DR on peripheral blood neutrophils 48 h after administration of rhIFN-γ (a) By setting a cut-off for HLA-DR induction at > 10% positive neutrophils HLA-DR responders (n = 22) were distinguished from HLA-DR nonresponders (n = 20). Expression of (b) CD64 and (c) CD74 on neutrophils was not different among HLA-DR responders and nonresponders after rhIFN-γ application (P = 0·9).

CD64 was induced by rhIFN-γ on neutrophils of both HLA-DR responders (n = 22; median Δ%CD64 75, IQR 37–88) and nonresponders (n = 20; Δ%CD64 80, IQR 44–90). Expression of CD64 was not different between HLA-DR responders and nonresponders at baseline (P = 0·9) as well as after application of rhIFN-γ (P = 0·9; Fig. 1). No correlation between the expression of HLA-DR and CD64 on neutrophils could be discerned within the entire study cohort (r = 0·08, P = 0·55). At baseline neutrophils from patients (n = 10) and healthy donors (n = 5) were virtually devoid of invariant chain (CD74) immunoreactivity. After in vivo rhIFN-γ administration CD74 was up-regulated in responders (n = 5, median 52%, IQR 35–68) and nonresponders (n = 5, median 51%, IQR 35–56) to a similar level (P = 0·7; Fig. 1).

During repeated cycles of rhIFN-γ therapy HLA-DR expression pattern on peripheral blood neutrophils was conserved amongst individuals (Fig. 2).

Fig. 2.

Fig. 2

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In vivo expression of HLA-DR on neutrophils during representative, repeated treatment cycles with rhIFN-γ. Three selected responders are shown (a–c) and three nonresponders (d–f). At each treatment cycle percentage of HLA-DR expressed at baseline (b) and post-treatment (p), i.e. 48 h after rhIFN-γ administration, are shown.

Serum IL-3 levels, GM-CSF levels, and peripheral blood neutrophil counts

Whether IFN-γ induced cytokines known to up-regulate HLA-DR expression in vivo, GM-CSF and IL-3 serum levels were measured during rhIFN-γ therapy. At baseline, GM-CSF was detectable in 2 of 42 patients, while IL-3 in none. Serum concentrations of GM-CSF and IL-3 did not change during treatment in those patients. Absolute peripheral blood neutrophil counts significantly decreased from 4·7 × 109/l (IQR 4·1–5·8) at baseline to 3·3 × 109/l (IQR 2·8–4·2, P = 0·002) after in vivo rhIFN-γ administration. No change in the ratio of band to segmented neutrophil forms was noted. Blasts or other immature myeloid forms were absent from blood smears.

In vivo kinetics of HLA-DR and CD64 expression on neutrophils and of serum IFN-γ levels after rhIFN-γ application

In three selected HLA-DR responders as well as in three nonresponders expression of HLA-DR and CD64 on neutrophils, and in parallel serum levels of IFN-γ were monitored over 72 h after rhIFN-γ administration. In responders, HLA-DR expression on neutrophils start to increase within the first 10 h and reached a maximum at 48 h (Fig. 3a). In nonresponders, HLA-DR expression on neutrophils ranged from undetectable to less than 4% positive cells at all time points investigated. Expression of CD64 also increased after rhIFN-γ application and reached a peak value between 24 and 48 h (Fig. 3b). Induction of CD64 on neutrophils did not differ between HLA-DR responders and nonresponders.

Fig. 3.

Fig. 3

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In vivo expression of (a) HLA-DR and (b) CD64 on neutrophils and (c) serum levels of IFN-γ in patients (n = 6) before as well as 4, 10, 24, 48 and 72 h after rhIFN-γ application. Responders [R] are shown as solid, nonresponders [NR] as dotted lines.

Baseline serum IFN-γ concentrations were below the detection limit of 3 pg/ml. Levels peaked 4–10 h after subcutaneous administration of rhIFN-γ (Fig. 3c). Serum IFN-γ was still detectable in 4 of 6 patients at 24 h and returned to baseline levels in all patients after 48 h. Calculation of areas under the curve for serum IFN-γ revealed no difference between HLA-DR responders and nonresponders (P = 0·4).

In vitro kinetics of HLA-DR and CD64 expression on isolated neutrophils stimulated by rhIFN-γ

In vitro kinetic studies of HLA-DR and CD64 were performed with neutrophils from the same HLA-DR responders and nonresponders who underwent the detailed in vivo analysis. Neutrophils isolated from responders at baseline responded to in vitro rhIFN-γ exposure with a time- and dose-dependent up-regulation of HLA-DR. As seen in vivo, a maximum expression of HLA-DR was observed later than 24 h of IFN-γ stimulation (Fig. 4a). In nonresponders, hardly any HLA-DR immunoreactivity could be discerned (Fig. 4b). CD64 was induced both in HLA-DR responders (Fig. 4c) and nonresponders (Fig. 4d) in a dose-dependent fashion with peaks at 24 h after rhIFN-γ stimulation. Donor dependent HLA-DR regulation could be recapitulated on isolated neutrophils from 10 healthy donors stimulated in vitro with 100 U/ml for 44 h. 6/10 healthy subjects were rated as HLA-DR responders, 4/10 as nonresponders (Fig. 4e).

Fig. 4.

Fig. 4

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In vitro regulation of HLA-DR (a, b) and CD64 expression (c, d) on isolated neutrophils from patients stimulated with rhIFN-γ (0·1, 1, 10, 100 U/ml) for 24 and 44 h. Responders (R; n = 3) are shown in the upper, nonresponders (NR; n = 3) in the lower panel. (e) Regulation of HLA-DR and CD64 on neutrophils isolated from healthy donors (HD) after in vitro stimulation with rhIFN-γ (100 U/ml) for 44 h recapitulated results of donor dependent induction from patients as shown for 6 representative subjects. (f) In patients rated as HLA-DR responders and nonresponders HLA-DR regulation on neutrophils was further investigated by in vitro incubation of cells with rhIFN-γ (100 U/ml) or GM-CSF (200 U/ml) or a combination of both cytokines for 44 h. In vitro kinetic studies were performed with neutrophils purified to>98%. Viability of neutrophils was>70% in all experiments after 44 h.

In vitro exposure to GM-CSF for 44 h resulted in the induction of HLA-DR on enriched neutrophils both from the patients classified as HLA-DR responders and from those categorized as nonresponders. Co-incubation of IFN-γ and GM-CSF increased HLA-DR expression in a synergistic manner even in HLA-DR nonresponders (Fig. 4f).

Transcription of HLA-DR mRNA in neutrophils after in vivo rhIFN-γ application

HLA-DR α chain transcripts could not be amplified from purified neutrophils of healthy donors (n = 3). Twenty-four hours after in vivo rhIFN-γ application HLA-DR α chain mRNA was detectable from neutrophils of responders (n = 3) and nonresponders (n = 3; Fig. 5).

Fig. 5.

Fig. 5

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Reverse transcription-polymerase chain reaction (RT-PCR) analysis of HLA-DRα (upper lane) and α-actin mRNA (lower lane) from purified neutrophils of healthy donors (HD), and patients after in vivo application of rhIFN-γ. HLA-DRα mRNA was detectable from neutrophils of responders (R) and nonresponders (NR).

Detection of HLA-DR stable dimers from neutrophils by Western blot analysis

In neutrophils of healthy donors and patients prior to rhIFN-γ administration HLA-DR αβ dimers could not be detected ex vivo by immunoblot analysis. HLA-DR αβ dimers became clearly detectable in neutrophils from healthy donors in vitro stimulated with IFN-γ and GM-CSF. In responders (n = 3), Western blot analysis of in vivo IFN-γ stimulated neutrophils was positive for HLA-DR αβ dimers whereas nonresponders remained negative (n = 3). In all Western blot experiments immunoreactivity to mAb L243 disappeared in heat denaturating conditions (Fig. 6).

Fig. 6.

Fig. 6

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(a) Ex vivo detection of HLA-DR αβ-dimers from peripheral blood cells from healthy donors. PBMCs, neutrophils, and neutrophils after in vitro stimulation with rhIFN-γ plus rhGM-CSF for 44 h served as controls. Cell lysates were treated at room temperature (RT) or denatured by boiling at 96°C (B). (b) Ex vivo detection of HLA-DR dimers in lysates of neutrophils from responders and nonresponders at baseline and 48 h after in vivo rhIFN-γ application.

Expression of HLA-DR and CD64 on peripheral blood monocytes after in vivo application of rhIFN-γ

In vivo, rhIFN-γ enhanced the level of HLA-DR (P < 0·001) and CD64 (P < 0·001) expression on monocytes (n = 42; Table 1). A more pronounced increment of HLA-DR on monocytes (ΔHLA-DR) was observed in HLA-DR responders than in nonresponders (P < 0·001). When we plotted the ΔHLA-DR of in vivo treated neutrophils against the ΔHLA-DR of monocytes, a striking correlation between the two data sets was obtained (r = 0·75, P < 0·0001; Fig. 7), together suggesting a concordant regulation of HLA-DR on neutrophils and monocytes. No difference in IFN-γ induced expression of CD64 on monocytes (P = 0·2) could be discerned between HLA-DR responders and nonresponders (Table 1).

Table 1.

Expression of CD64 and HLA-DR on monocytes in vivo

HLA-DR CD64
n Baseline Post rhIFN-γ Baseline Post rhIFN-γ
All patients 42 169 (99–223) 286 (199–401) 139 (100–168) 270 (225–308)
HLA-DR responders 22 188 (107–231) 329 (289–418) 127 (108–153) 272 (246–308)
HLA-DR nonresponders 20 117 (58–218) 181 (107–250) 149 (86–184) 262 (206–309)
P-value = 0·16 <0·001 = 0·33 = 0·54
Healthy donors 47 198 (164–233)  84 (60–104)
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Data are presented as median (IQR) of median channel fluorescence intensities. P -value relates to the differences between HLA-DR responders and nonresponders both at baseline and after treatment with rhIFN-γ.

Fig. 7.

Fig. 7

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Plotting ΔHLA-DR surface expression of in vivo rhIFN-γ treated neutrophils vs. ΔHLA-DR of monocytes from all patients (n = 42). Vertical, dotted line at ΔHLA-DR neutrophils delimits HLA-DR nonresponders (left) from HLA-DR responders (right).

DISCUSSION

Human peripheral blood neutrophils do not express MHC class II antigens constitutively. The present study is the first comprehensive and conclusive description of donor dependent HLA-DR regulation on neutrophils in vivo, a finding which has so far been solely described in vitro with small numbers of healthy donors. The demonstrated correlation of in vivo and in vitro results confirms the biological significance of our observations. Our data suggest that (post) translational, but not transcriptional events are operative in a donor dependent fashion in IFN-γ induced HLA-DR regulation in neutrophils. For the first time, we were able to show that in vivo IFN-γ mediated donor dependent HLA-DR regulation on neutrophils is correlated with that on monocytes.

Previously, we have described the induction of HLA-DR on neutrophils in peripheral blood of four patients with metastatic hypernephroma after in vivo application of rhIFN-γ[16]. Based on this initial observation we extended our investigations to a large cohort. To illustrate a possible heterogeneity of IFN-γ mediated HLA-DR induction on neutrophils we stratified patients as HLA-DR nonresponders and HLA-DR responders by setting a cut-off for HLA-DR induction at > 10% positive neutrophils. IFN-γ mediated expression of HLA-DR on neutrophils in vivo varied markedly among patients with an even distribution among the two strata. The group of HLA-DR nonresponders (n = 27) included also five individuals lacking any surface expression of HLA-DR. The reproducibility of donor dependent HLA-DR expression patterns on neutrophils was confirmed during repeated cycles of rhIFN-γ therapy. The differential expression of HLA-DR on neutrophils in responders vs. nonresponders might be due to a cell autonomous regulatory mechanism, which is supported by the concordance of results obtained in in vivo and in vitro kinetic studies performed on three selected HLA-DR responders and nonresponders. It could be hypothesized that the phenomenon described in this study could result from confounding variables due to the underlying malignant disease state. However, the donor dependent IFN-γ mediated HLA-DR regulation in isolated neutrophils from healthy donors in vitro refutes this possibility. We even suggest that discrepant results reported on the in vitro regulation of MHC class II molecules via IFN-γ in studies with small numbers of healthy donors originate from the mechanisms described herein [12,19,20,27,28].

Different expression levels of HLA-DR on neutrophils during rhIFN-γ treatment could be explained by donor dependent pharmacokinetics of IFN-γ or by diverse induction of secondary mediators such as GM-CSF and IL-3, both known to regulate MHC class II molecules on these cells [12,13,17]. Serum levels of IFN-γ monitored during time kinetics were not different between HLA-DR responders and nonresponders. In addition, serum levels of GM-CSF and IL-3 were undetectable or unchanged during the course of therapy. Neither a change in the ratio of band to segmented forms nor the appearance of immature myeloid forms in blood smears could be detected. Together, all these observations further indicate that HLA-DR expression on neutrophils is cell autonomous rather than mediated via different IFN-γ pharmacokinetics or induction of GM-CSF and IL-3.

To control IFN-γ signalling as potential cause of donor dependent variations in HLA-DR expression on neutrophils, we investigated the high affinity Fcγ-receptor (CD64), which is tightly regulated by IFN-γ due to binding of STAT1 to the IFN-γ response region in its promoter [29]. This regulation shares similarities to the transcriptional activation of class II transactivator (CIITA) in its promoter IV region by STAT1 [6]. During in vivo and in vitro kinetic studies CD64 was equally induced in both HLA-DR responders and nonresponders. Similarly, no differences in up-regulation of cell-surface MHC class I molecules and enhancement of cell viability in neutrophils among the HLA-DR response groups could be discerned (data not shown). Therefore, IFN-γ signalling appears functional in HLA-DR nonresponders, and pronounced donor dependent induction of HLA-DR in neutrophils is not attributable to general heterogeneity of IFN-γ responsiveness.

In line with functional IFN-γ signalling, IFN-γ induced HLA-DR α-chain was expressed at the transcriptional level in neutrophils of HLA-DR responders and patients lacking HLA-DR cell surface expression. As shown recently, by using our established technique for high purification of neutrophils, we can exclude the possibility that PCR signals for HLA-DR mRNA derive from other cell types than neutrophils [26]. Furthermore, IFN-γ mediated induction of invariant chain (CD74) in HLA-DR nonresponders demonstrates that CIITA, which controls HLA-DR and CD74 in a coordinated manner, is transcriptionally active at promoter sites of both genes [9]. Therefore, lack of HLA-DR induction upon stimulation with IFN-γ in neutrophils might not emanate from transcriptional blockade. This contrasts the mechanims of infectious agents, cytokines and drugs, which have been shown to impair MHC class II antigen expression by inhibiting CTIIA induction [30–35].

In order to elucidate whether mRNA transcripts of HLA-DR are translated in neutrophils of nonresponders lacking any surface expression, we searched for possible intracellular pools of MHC class II αβ-dimers. However, intracellular HLA-DR molecules could not be detected with mAb L243 in these cells. This could be explained by translational or post-translational control mechanisms. In fact, mutant or incorrectly folded MHC class II complexes have been described to be rapidly degraded, which results in deficient protein expression [36]. We can not exclude structural changes of HLA-DR α or β chains in nonresponders since L243 detects a conformational, nonpolymorphic HLA-DR epitope composed of both subunits. Attempts to detect HLA-DR monomers by employment of different α- or β-specific mAbs in immunoblot analysis yielded equivocal results, most probably due to neutrophils’ rich content of proteases (data not shown). Nevertheless, structurally altered α or β chains due to mutations are an unlike explanation for the HLA-DR nonresponsiveness since in vitro stimulation of neutrophils from nonresponders with GM-CSF resulted in significant cell surface expression of HLA-DR. GM-CSF induced expression of HLA-DR on neutrophils was potentiated by costimulation with IFN-γ even in nonresponders.

We suggest that translational or post-translational events, rather than polymorphisms or mutations of the HLA-DR subunits themselves mediate donor dependent IFN-γ induced HLA-DR expression in neutrophils. This may involve regulation of chaperones or proteases [10]. The fact that IFN-γ mediated induction of HLA-DR on monocytes correlates with donor dependent responsiveness in neutrophils points to a general principle of heterogeneous MHC class II antigen regulation in humans.

The inclusion of rhIFN-γ in therapeutic strategies directed against hypernephroma has been associated with favourable outcomes and the induction of MHC class II antigens has been considered as a rationale for its therapeutic use [22]. In the present study correlations of HLA-DR expression and survival were not performed due to the potential bias from the biological effects of sequentially administered rhIL-2 after rhIFN-γ application in our patients. A key role of HLA-DR expression in monocytes is well documented in various clinical settings. Low HLA-DR expression as a result of monocyte deactivation during sepsis is associated with a poor outcome. In this scenario, treatment with rhIFN-γ leading to restoration of deficient HLA-DR expression has been shown to result in clearance of sepsis [37]. Less is known about the functional relevance of HLA-DR expression on neutrophils. In vitro HLA-DR expressing neutrophils mediate superantigen-driven activation of CD4+ T cells and the magnitude of the resulting T cell responses correlates with the level of HLA-DR expression [19]. Since neutrophils are a predominant cell type in various inflamed tissues HLA-DR expression on these cells could modulate diseases mediated by superantigen. Whether neutrophils could function as APCs for soluble antigens such as tetanus toxoid is controversially discussed [19,20]. Currently, we address the biological role of donor dependent IFN-γ induced HLA-DR expression on neutrophils within the scope of a further prospective in vitro and in vivo study.

Our finding of a strictly donor dependent, IFN-γ induced regulation of HLA-DR on neutrophils highlight these cells as a unique model system to define novel elements involved in MHC class II antigen assembly. Furthermore, elucidation of heterogenous IFN-γ responsiveness in respect to HLA-DR induction may contribute to our understanding of individual susceptibilities and courses of various diseases including malignancies, autoimmunity and infection.

Acknowledgments

The authors thank Claudia Polli, Matthew Allen, Markus Wolschek and Schaker Zakeri for technical assistance as well as Birgit Winkler for critical reading of the manuscript. This work was supported by the Kommission Onkologie der Medizinischen Fakultät der Universität Wien and Centre of Molecular Medicine of the Austrian Academy of Sciences.

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