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Rheumatology Advance Access originally published online on March 23, 2007
Rheumatology 2007 46(6):934-937; doi:10.1093/rheumatology/kem039
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Neutrophils of healthy subjects with a history of reactive arthritis show enhanced responsiveness, as defined by CD11b expression in adherent and non-adherent whole blood cultures

K. Kuuliala, A. Orpana1, M. Leirisalo-Repo2 and H. Repo3

Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, 1Department of Clinical Chemistry and Department of Medical Genetics, Laboratory Diagnostics, 2Division of Rheumatology and 3Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital, Helsinki, Flinland

Correspondence to: Krista Kuuliala, Haartman Institute, Department of Bacteriology and Immunology, P.O. Box 21 (Haartmaninkatu 3) FIN-00014, University of Helsinki, Finland. E-mail: krista.anttonen{at}helsinki.fi


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. To study innate immune responsiveness of HLA-B27 positive subjects recovered from Yersinia-triggered reactive arthritis (B27 + ReA+).

Methods. Whole blood samples from 15 B27 + ReA+, 15 B27 + ReA– and 15 B27 – ReA– subjects were heparinized, aliquoted and (i) kept at 0°C to preserve constitutive cell surface marker status, or (ii) cultured with or without bacterial lipopolysaccharide (LPS) supplement, in adherent and non-adherent conditions at 37°C for 4 h. Neutrophil surface expression of CD11b, CD14 and CD16 was quantified flow cytometrically, and compared between the subject groups using Jonckheere–Terpstra test.

Results. The B27 + ReA+ group showed significantly higher CD11b levels than the B27 – ReA– group on non-adherent neutrophils cultured with LPS as 100 pg/ml (P = 0.027), 10 ng/ml (P = 0.048) or 1 µg/ml (P = 0.024), or on adherent neutrophils without LPS supplement (P = 0.040). CD14 and CD16 expression on cultured neutrophils and constitutive expression of all three markers were comparable between the groups.

Conclusions. Enhanced neutrophil reactivity observed may exacerbate innate immune inflammation in HLA-B27 positive ReA patients.

KEY WORDS: ReA, Innate immunity, CD11b, Adhesion, Lipopolysaccharide, HLA-B27


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Reactive arthritis (ReA) is a sterile joint inflammation following a mucosal infection, typically caused by Gram-negative bacteria, such as Chlamydia, Yersinia, Salmonella, Campylobacter or Shigella. Mainly, ReA affects human leukocyte autigen (HLA)-B27 positive individuals [1]. The pathogenesis of ReA is unclear, but may involve aberration(s) in adaptive [2] and/or innate [3] immune responses to the arthritogenic microbes. Neutrophils and monocytes, the phagocytic innate immune cells, can be activated by inflammatory stimuli, such as bacterial lipopolysaccharide (LPS), which is recognized by the CD14/TLR (Toll-like receptor) 4/MD-2 -complex [4] on phagocyte surface. Upon neutrophil activation, CD14 is increased on the plasma membrane from cytoplasmic granules [5] and on prolonged LPS stimulation, by de novo synthesis [6]. Expression of CD11b/CD18 (Mac-1), a ß2 integrin which mediates adhesion [7] and regulates several cellular functions [8, 9], also increases [10, 11]. Elevated CD11b expression has been used in clinical studies as a phagocyte activation marker [12–16]. CD16 (low-affinity IgG receptor), which may contribute to inflammatory tissue injury [17], initially increases on LPS-stimulated neutrophils, but decreases later along with commitment to apoptosis [6].

We studied ReA-associated innate immune function by exposing blood samples from healthy subjects with previous Yersinia-triggered ReA to LPS. The experiments were performed ex vivo in adherent and non-adherent conditions to mimic the intravascular milieu where activated phagocytes may adhere to the endothelium or continue circulating [7]. Phagocyte activation was evaluated by CD11b, CD14 and CD16 expression of immunostained samples flow cytometrically. The present communication comprises neutrophil activation results, also correlated with monocyte function results reported previously [18].


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Subjects
Forty-five Finnish volunteers participated, as described in detail previously [18]. Fifteen HLA-B27 positive subjects had had Yersinia-triggered ReA with complete recovery (B27 + ReA+ group) and participated in clinical studies of acute ReA [19] and disease outcome [20]. Fifteen of the healthy reference subjects were HLA-B27 positive (B27 + ReA– group) and fifteen HLA-B27 negative (B27 – ReA– group). The study protocol was approved by the local ethics committee and the subjects gave their informed consent.

Reagents
We used: heparin (Lövens, Ballerup, Denmark); Dulbecco's phosphate buffered saline (PBS) and RPMI 1640 medium (Life Technologies Ltd., Paisley, UK); Escherichia coli O111:B4 LPS (Sigma, St Louis, Missouri, USA); AB serum (Finnish Red Cross, Helsinki, Finland); FITC (fluoresceine isothiocyanate)-linked mouse anti-CD11b mAb (IgG1, clone BEAR1), PC5 (phycoerythrin-CY5)-linked anti-CD16 mAb (IgG1, clone 3G8) and irrelevant mouse mAb IgG1 (clone 679.1Mc7) (Immunotech, Marseille, France); RPE (R-phycoerythrin)-linked anti-CD14 mAb (IgG2a, clone TÜK4) and irrelevant mAb (IgG2a, clone DAK-GO5) (DAKO A/S, Glostrup, Denmark); FACS lysing solution and QuantiBRITE PE standards (Becton Dickinson, San Jose, California, USA).

Blood samples
One 3 ml heparinized blood sample from each subject was cooled immediately and aliquoted for cultures or flow cytometry, as described previously [18].

Methods
Whole blood culture assay
LPS stock solution (400 µg/ml PBS) was stored at –20°C. One set of heparinized blood aliquots (100 µl each) was cultured in polypropylene test tubes (No. 352063, Becton Dickinson) which do not support adhesion, in RPMI 1640 (800 µl) and LPS (100 µl) as 100 pg/ml, 10 ng/ml, or 1 µg/ml, or in RPMI 1640 alone (900 µl). Concomitantly, another aliquot set was cultured in adhesion-supporting cell culture wells (No 3515, Corning Inc, Corning, NY, USA). As strong adhesion to endothelium initiates an entirely new stage in the leucocyte life course, adhesion-mediated messages may overwhelm weaker simultaneous signals, e.g. low LPS concentrations. Thus, the samples in wells were cultured in RPMI 1640 and LPS as 1 µg/ml, or in RPMI 1640 alone. The tubes and wells were incubated at 37°C in 5% CO2 for 4 h (sufficient time to increase neutrophil surface marker expression notably [6, 7]).

After incubation, cells were pelleted by centrifugation, re-suspended in ice-cold AB serum, aliquoted (2 x 25 µl) into polystyrene tubes (No. 352054, Becton Dickinson) and retained at 0°C until staining.

Leucocyte surface marker staining
Three-colour flow cytometry was used as described previously [18]. Briefly, pre-titrated amounts of FITC-labelled anti-CD11b, RPE-labelled anti-CD14 and PC5-labeled anti-CD16 antibodies, or irrelevant isotype-specific antibodies were used. After staining at 0°C for 20 min, erythrocytes were lysed and data acquisition was performed within 4 h.

Flow cytometry
A FACSort flow cytometer (Becton Dickinson) and CellQuest software were used for data acquisition as described elsewhere [21]. The data were analysed using QuantiCalc software (Verity Software House, Topsham, Maine, USA). Neutrophil CD11b levels, expressed as relative fluorescence units (RFUs), were obtained by plotting the FITC intensity (CD11b) against the PC5 intensity (CD16) of the collected events. Monocyte contamination was excluded by electronic gating based on the high CD16 expression on neutrophils in comparison with monocytes. The number of PE-linked anti-CD14 molecules equals to each neutrophil's CD14 antibody binding capacity (ABC). ABC medians were determined by creating a fluorescence intensity plot PE (CD14) vs PC5 (CD16) [21]. CD16 levels are given as RFUs.

TNF determination
Determination of tumour necrosis factor (TNF) levels from whole blood culture supernatants is described in [18].

Data analysis
LPS dose dependence of CD11b, CD14 and CD16 levels of all subjects’ (n = 45) neutrophils and the effect of adhesion vs LPS doses were evaluated by Friedman test. In comparison of two different conditions only, Wilcoxon signed rank test was used.

Neutrophil CD11b, CD14 and CD16 levels were compared between the three subject groups (each n = 15) by Jonckheere–Terpstra test for linear trend, which was chosen because B27 + ReA+ subjects had both HLA-B27 (genetic factor predisposing to ReA) and a history of the disease, B27 + ReA– subjects had the genetic factor only, and B27 – ReA– subjects had neither.

Monocyte TNF production in whole blood, stimulated by adherence and/or LPS as 1 µg/ml [18], or by LPS as 10 ng/ml or 100 pg/ml, was compared between the subject groups by Jonckheere-Terpstra test. CD11b, CD14 and CD16 levels, if found significantly different between the groups, were correlated with the TNF levels using Spearman correlation.

A P-value <0.05 was considered statistically significant. Dunn correction was used to adjust for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Constitutive expression
Constitutive CD11b, CD14 and CD16 levels on whole blood neutrophils were compared with those of blood samples incubated in non-adherent conditions without LPS supplement at 37°C for 4 h. Constitutive level medians (ranges) were significantly lower [81 (43–164) vs 433 (164–1241) RFUs for CD11b, ABC 262 (171–567) vs 505 (389–1073) for CD14 and 1433 (389–3786) vs 2329 (770–5094) RFUs for CD16] (P-values <0.001, Wilcoxon test).

Effect of assay conditions
LPS dose
CD11b levels of non-adherent neutrophils showed a significant LPS dose-dependent trend (Fig. 1, Table 1; P < 0.001, Friedman test with Dunn correction). CD14 showed a corresponding trend, ABC medians (ranges) being 505 (389–1073), 631 (405–1505), 758 (541–1610) and 765 (535–1626) for LPS as 0, 100 pg/ml, 10 ng/ml and 1 µg/ml (P < 0.001), respectively, albeit the levels obtained with the two highest LPS concentrations were comparable (P = 1.000, Dunn correction). An LPS dose-dependent trend was not observed in CD16 levels, RFU medians (ranges) by ascending LPS concentration being 2329 (770–5094), 2763 (88–5280), 2618 (1000–4371) and 2414 (820–5094) (P = 0.853).


Figure 1
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  		FIG. 1. Expression levels of CD11b on peripheral blood neutrophils after a 4-h incubation in various concentrations of LPS at 37°C. Subjects comprise 15 HLA-B27 positive subjects recovered from Yersinia-triggered reactive arthritis (B27 + ReA+), and 15 HLA-B27 positive (B27 + ReA–) and negative (B27 – ReA–) subjects with no history of ReA. Each box denotes medians with 25th to 75th percentiles, whiskers showing minimum and maximum values, excluding outliers (i.e. values between 1.5 and 3 box lengths outside the box, denoted by circles) and extremes (i.e. values more than 3 box lengths outside the box, denoted by asterisks). Subject groups were compared using Jonckheere–Terpstra test for linear trend, and adjusted for multiple comparisons using Dunn correction.

 

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  		TABLE 1. Correlation between neutrophil CD11b levels and monocyte TNF production levels, determined in whole blood samples after a 4-h incubation with LPS at 37°C

 
Adhesion
CD11b (Fig. 1), CD14 [ABC 607 (405–1520)] and CD16 [2595 (770–5186) RFUs] levels of adherent neutrophils with no administrated LPS were comparable with those of non-adherent neutrophils with LPS as 10 ng/ml for CD11b, 100 pg/ml for CD14 and 0, 100 pg/ml, 10 ng/ml and 1 µg/ml for CD16 (Friedman test with Dunn correction).

Adherent neutrophils with LPS as 1 µg/ml showed higher CD11b (Table 1, Fig. 1), and lower CD14 [ABC 562 (364–1277)] and CD16 [1731 (777–4532) RFUs] levels (P-values <0.001, Wilcoxon test) than the respective non-adherent neutrophils.

Comparison of the subject groups
On non-adherent neutrophils with all LPS concentrations studied, and on adherent neutrophils without LPS supplement, CD11b expression showed a trend: the B27 + ReA+ group had the highest and B27 – ReA– group the lowest levels (Fig. 1, Jonckheere–Terpstra test with Dunn correction). Neither the constitutive levels of the three markers nor the CD14 or CD16 levels of cultured neutrophils differed between the subject groups (Jonckheere–Terpstra test, data not shown).

TNF production
LPS-stimulated TNF production by monocytes in whole blood was comparable in the groups (Table 1, Jonckheere–Terpstra test).

When stimulated with LPS as 100 pg/ml, a positive correlation was observed in all subject groups between neutrophil CD11b levels and monocyte TNF production in whole blood (Table 1, Spearman correlation).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
The results show that when stimulated with LPS or adhesive conditions for 4 h, significantly higher CD11b levels on neutrophils, suggesting enhanced neutrophil activity, occur in B27 + ReA+ than B27 – ReA– blood samples.

Bacterial LPS may persist in ReA patients [22]. The CD11b expression differences might derive from aberrations in LPS signalling pathways, e.g. functional TLR4 polymorphisms [23] since intracellular signalling from TLR4 leads to CD11b up-regulation on neutrophils [11], or at the level of CD11b/CD18 since this is a ß2-integrin with important signalling capabilities. Also, LPS-stimulated monocyte TNF production did not differ between the subject groups, suggesting that the differences in CD11b levels cannot be explained by aberrations in TNF production or TNF-mediated signalling in neutrophils. Although the explanation remains unknown, it may involve abnormal priming of neutrophils by LPS [24, 25]. Neutrophils of individuals with previous Yersinia arthritis may be primed, as evaluated by enhanced oxygen radical production [26]. Our findings are unlikely to signify an ongoing cryptic infection, because the CRP levels [18] and constitutive neutrophil surface marker levels were comparable in the subject groups.

Neutrophil CD11b levels exhibited a trend: B27 + ReA + > B27 + ReA – > B27 – ReA–. Consequently, previous ReA and HLA-B27 positivity may be contributing factors for the difference to appear. Recent findings provide evidence that misfolded B27 polypeptides cause stress in the endoplasmic reticulum [27], which may affect CD11b/CD18 transport [10] or de novo synthesis [11].

In systemic inflammatory conditions, activated neutrophils may exist in the circulation [7]. We therefore designed our in vitro whole blood assay to include both adherent and non-adherent conditions. Neutrophil CD11b expression levels were comparable with LPS as 10 ng/ml in non-adherent tubes and without LPS supplement in adherent wells. LPS as 10 ng/ml or less may activate phagocytes via the classical CD14/TLR4/MD-2 pathway, whereas 1 µg/ml or more may signal via CD11b/CD18 [28]. This may partly explain our present finding that low LPS levels (100 pg/ml and 10 ng/ml) in non-adherent conditions increased CD14 expression, whereas 1 µg/ml in adherent wells (our maximum stimulus) depressed CD14 expression. CD16 was also decreased by the maximum stimulus. On prolonged LPS exposure, neutrophil CD16 expression has been reported to decrease coinciding with apoptosis induction [6]. In vivo, apoptosis apparently prevents excessive neutrophil activation [6] and tissue damage [17]. Taken together, our method provides a means to study the regulation of blood leucocyte activation in inflammatory conditions by applying flow cytometric techniques [29].

In conclusion, these preliminary results show that when stimulated by LPS or adherence, neutrophils of HLA-B27 positive subjects recovered from ReA show significantly higher responsiveness, evaluated by CD11b expression, than those of HLA-B27 negative reference subjects. Such an innate immune reactivity may play a role in the development of ReA.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We wish to thank Eine Virolainen and Maija Peltoperä for skilful technical assistance. The study was supported by grants from Finska Läkaresällskapet and Finnish Cultural Foundation to KK.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 28 March 2006; revised version accepted 24 January 2007.
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