Reduced numbers of peripheral blood CD27⁺ IgD^– memory B cells in patients with aggressive periodontitis

Introduction

Aggressive periodontitis (AgP) represents a type of periodontitis with a rapid rate of disease progression that affects people who, in most cases, appear otherwise healthy. Epidemiological surveys have shown that the prevalence rates for AgP vary from 0.1% to 17.6%, whereas the disease is less common in Caucasian populations and more frequent in India and Africa [1, 2]. AgP is a multifactorial disease that tends to have a familial aggregation [3]. Pathogenic bacteria like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Fusobacterium nucleatum represent the primary triggers in the etiology of periodontitis [4]. However, most of these bacteria are part of the normal oral microflora. Thus, additional host susceptibility factors such as single-nucleotide polymorphisms within inflammatory genes, epigenetic regulation, and also smoking behaviour, race, age, and the environment may play a role [5]. The aim of this investigation was to elucidate a connection between periodontal condition in patients with AgP and their possible state of immunodeficiency.

Patients and methods

Immunological investigation

Eighteen millilitres of peripheral blood was collected to analyse the composition of lymphocyte subpopulations and the peripheral B cells by flow cytometry (FCM) and to stain T cells intracellularly for IFNγ and IL-4 (ICS) [9–11]. Furthermore, we determined the level of serum IgM, IgG, and IgA and their Ig-subclasses and cultured B cells for 7 days in the presence of IL-10 and anti-CD40 mAb to analyse their corresponding IgG and IgA production (Figure 1). The release of IFNγ, IL-10, IL-1β, IL-8 (CXCL8), and RANTES (CCL5) by peripheral blood mononuclear cells (PBMC) upon stimulation with either phytohaemagglutinin, lipopolysaccharide (LPS, E. coli 055:B5), tetanus toxoid, pneumococcal cell wall polysaccharide (Streptococcus pneumoniae strain), or mumps virus lysate (Enders strain) was investigated using single-cell enzyme-linked immunospot (ELISPOT) technique as previously described and is shown in Figure 1 [12]. All data from FCM, intracellular staining (ICS), and ELISPOT analyses were subjected to heatmap row-normalization and visualization using Heatmap Builder v1.1 (E. A. Ashley, J. M. Spin, and C. Watt: Quertermous Lab, Stanford University, Stanford, CA, USA). Blood samples for ELISPOT assay were taken in 8 of 10 patients. In addition, eight healthy blood donors who did not differ in terms of age and sex were included as experimental controls.

Figure 1:

Heatmap visualization of the patient’s immunological findings obtained by flow cytometry, intracellular cytokine staining, and ELISPOT assays.

Results

In general, dental and radiographic examination revealed increased clinical attachment levels (mean 3.7 mm, normal=0 mm) corresponding to an extensive radiographic bone loss and PD (mean 3.6 mm, normal ≤2 mm) in all patients, as indicated in Figure 2 and Table 1. Major periodontal pathogens were detected by microbiological culture of subgingival plaque samples, as shown in Table 1. The patient’s clinical reports delivered no evidence for increased susceptibility to infection or frequent need for antibiotic treatment. Two of the 10 patients had received antibiotics within a former periodontal therapy 22 months before taking blood samples for ELISPOT assay.

Figure 2:

Clinical aspect and Orthopantomograph image of a representative patient diagnosed with AgP.

(A) and (B): Photographic documentation of the clinical aspect; arrows point at displaced tooth and suppurations in several pockets. (C) Orthopantomograph image; arrows point at severe periodontal destruction sites with radiographic bone loss.

FCM analysis revealed a substantial reduction of IgM^– IgD^– CD27⁺ switched memory B cells in the peripheral blood of all patients presented herein, whilst the proportions of naïve, IgM⁺ memory, transitional, and activated B cells were all normal, as well as the composition of other lymphocyte subpopulations (Figure 1). Serum levels of IgM, IgG, IgA, and subclasses were all normal (data not shown), and the amount of IgG and IgA produced upon in vitro differentiation of B cells was comparable to that of healthy individuals. The percentage of both intracellular IL-4⁺ CD4⁺ and CD8⁺ T cells was considerably decreased in the peripheral blood of all analysed patients, whilst the percentages of IFNγ⁺ T cells were normal (Figure 1). In the ELISPOT assay, highly elevated IL-1β and IL-8 (CXCL8) responses to LPS and pneumococcal cell wall polysaccharide (CWPS) were discovered in all 8 of 10 analysed patients (Figure 1). Furthermore, the baseline production of RANTES (CCL5) was found to be elevated in the PBMC of patients with AgP.

Discussion

Many primary immunodeficiency diseases clinically present with oral manifestations such as periodontal inflammation, including diseases of phagocytic deficiency or T- and B-cell deficiency, and are commonly caused by the impairment of oral defence mechanisms against candidiasis and herpetic or bacterial infections [13]. In this regard, our finding of severely reduced numbers of switched memory B cells in the peripheral blood of patients with AgP is of certain importance. Whilst IgM^– IgD^– CD27⁺ switched memory B cells are the main source for isotype-switched serum immunoglobulins, such as IgG, IgA, and IgE, their exclusive reduction is typically referred to the state of common variable immunodeficiency type I syndrome (CVID), according to the classification system proposed by Warnatz et al. [14], with an association between the percentage of switched memory B cells and the frequency of infectious complications. However, as serum levels of IgM, IgG, IgA, and subclasses were all normal and no increased susceptibility to infection or frequent need for antibiotic treatment was found in our patients with AgP, the diagnosis of CVID is very unlikely. We would rather propose that elevated monocytic secretion of both IL-1β and IL-8 and lowering of IL-4 mediated macrophage inhibition triggers a pro-inflammatory circle that attracts lymphocytes from the peripheral blood to local pockets of aggressive periodontitis. Besides their strong chemotactic effects on leukocytes, both IL-1β and IL-8 are known stimulators and regulators of B cells, promoting their proliferation and the synthesis of immunoglobulins.

In accordance with the results of a study by Fiebig et al. [15], our data show that the increased secretion of IL-1β is activation-dependant in terms of a hyper-responsive macrophage phenotype, implying that polymorphisms in the IL-1 gene cluster may not generally increase IL-1β production in patients with AgP. Whilst several IL-4 promoter and intronic polymorphisms have been shown to correlate with the onset of periodontitis and the patient’s serum levels of IL-4, little is known about their impact on the peripheral lymphocyte compartment in AgP [16]. IL-4 is a potent down-regulator of macrophage function and prevents the production of IL-1β, TNFα, and prostaglandins in response to activation of monocytes. As shown in agreement with our results from intracellular IL-4 staining, IL-4 serum levels are typically reduced in patients with AgP, most likely unleashing the unfavourable chemotactic effects of IL-1β and IL-8 [16]. Finally, Sigusch et al. [17] have shown that CD20⁺ B cells are present 13 times more frequently in the periodontal pockets of patients with AgP than in healthy controls, supporting our hypothesis of B-cell migration from the peripheral blood to active sites in AgP.

In summary, our data shed light on the potential interplay of IL-1β, IL-8, and IL-4 to trigger a pro-inflammatory circle that attracts lymphocytes to local pockets in aggressive periodontitis.