Cellular inflammatory response to persistent localized Staphylococcus aureus infection: phenotypical and functional characterization of polymorphonuclear neutrophils (PMN)

Abstract

Persistent, localized Staphylococcus aureus infections, refractory to antibiotic treatment, can result in massive tissue destruction and surgical intervention is often the only therapeutic option. In that context, we investigated patients with S. aureus-induced infection at various sites, apparent as either olecranon bursitis, empyema of the knee joint or soft tissue abscess formation. As expected, a prominent leucocyte infiltrate was found, consisting predominantly of polymorphonuclear neutrophils (PMN) (up to 75%) and to a lesser extent of T lymphocytes and natural killer (NK) cells. In line with their bactericidal capacity, PMN expressed the high-affinity receptor for IgG, CD64 and the lipopolysaccharide (LPS) receptor CD14; moreover, the oxygen radical production in response to the bacterial peptide f-MLP was enhanced, while chemotactic activity was greatly reduced. The more intriguing finding, however, was that a portion of PMN had acquired major histocompatibility complex (MHC) class II antigens and CD83, indicative of a transdifferentiation of PMN to cells with dendritic-like characteristics. Of note is that a similar transdifferentiation can be induced in PMN in vitro, e.g. by gamma interferon or by tumour necrosis factor alpha. Co-cultivation of transdifferentiated PMN with autologous T lymphocytes resulted in prominent T cell proliferation, provided that S. aureus enterotoxin A was added. Taken together, persistent S. aureus infection induces PMN to acquire characteristics of dendritic cells, which in turn might promote the local immune response.

Fig. 1

Analysis of surface antigens on polymorphonuclear neutrophils (PMN) of patient 1 by cytofluorometry: PMN were identified by CD66b expression; within that gate, expression of CD14, CD62L and CD64 was measured in blood samples taken immediately prior to surgery, collected intraoperatively from the lavage or from the opened bursa. For comparison, peripheral blood obtained 28 days following surgery is shown (the thick lines show binding the specific antibody; the thin lines show the respective isotype controls).

Fig. 2

Expression of surface antigens of polymorphonuclear neutrophils (PMN) of all the patients (n = 12): The data obtained by cytofluorometry (as shown in Fig. 1) were summarized as statistical Box-and-Whisker blots with the box containing 50% of the values. The horizontal bar represents the median. Each panel summarizes the data for one of the receptors. Shown is the normal range (derived from 20 donors) (open box); expression on PMN of the peripheral blood taken prior to surgery (striped box); of the lavage (black box); of the drainage fluid obtained at day 2 post-surgery (hatched box); and in the peripheral blood 2–4 weeks after surgery (vertical stripes). Differences between the mean values calculated for the respective group and the normal range were determined by a t-test for unpaired samples; P-values are given; n.d. indicates that the groups were not different; the values shown as p* refer to differences between cells of the peripheral blood taken prior to surgery and cells of the lavage.

Fig. 3

Expression of major histocompatibility complex (MHC) class II antigens and of CD83 on polymorphonuclear neutrophils (PMN): (a) with an antibody recognizing HLA-DP-DQ-DR, expression of MHC class II antigen was found on PMN from the lavage (middle panel), the drainage fluid (right panel) but not on cells derived from the peripheral blood (left panel) (the thick lines show the specific antibody; the thin lines the isotype controls; data for patient 2 are shown). Similarly, expression of CD83 was seen on PMN derived from the lavage or the drainage fluid, but not on peripheral blood PMN. (b) Data for all patients are summarized (as detailed in the legend for Fig. 2). (c) Reverse transcription-polymerase chain reaction (RT-PCR) was performed with PMN derived from the peripheral blood or from the lavage of the same patient (data for patient 4 are shown) with primers specific for either CD83, MHC class II or GAPDH as housekeeping gene.

Fig. 4

Superoxide production and chemotaxis of polymorphonuclear neutrophils (PMN). Upper panel: the superoxide production in response to phorbol myristate acetate (PMA) (black squares) or f-MLP (black circles) of PMN was measured isolated from the lavage or the blood of the patients taken immediately prior to surgery, or the blood of healthy donors (n = 20), respectively. Open circles show spontaneous superoxide production. Shown are the mean values ± s.d.; * indicates that the mean value is different when compared to that of the PMN derived from the blood of the patients (P = 0·025) or of the donors (P = 0·001). Lower panel: the chemotaxis towards C5a (black circles) or interleukin (IL)-8 (black squares) was tested of blood-derived PMN (taken immediately prior to surgery) or from lavage of the patients. For comparison, migration of healthy donors (n = 20) was tested (the open squares show the spontaneous random migration). Shown are the mean values ± s.d.; * indicates that the mean value is different when compared to that of the PMN derived from the blood of the patients (P = 0·020) or of the donors (P = 0·001), respectively.

Fig. 5

Proliferation of T lymphocytes using polymorphonuclear neutrophils (PMN) as antigen-presenting cells: (a) PMN derived from the lavage of patient 12 (L-PMN, 1 × 10⁵) were co-cultivated with either lavage-derived T cells (L-T cells) or T cells isolated from the peripheral blood (pT cells, 2 × 10⁴) (taken immediately prior to surgery) in the absence (two left columns) or presence (two right columns) of staphylococcal enterotoxin (SE) A (10 ng/ml); proliferation was measured by incorporation of [³H]-thymidine (cpm). Values represent the mean ± s.d. of 12 parallel wells. *Indicates that proliferation is different from that seen in the absence of SEA (P = 1·5 × 10⁻⁸); **proliferation of pT cells is enhanced compared to L-T cells (P = 1·2 × 10⁻⁸). (b) CD4 positive T cells (obtained from the lavage of patient 11) were cultivated in the presence of heterologous PMN and either SEA or SEB in the concentrations indicated; proliferation of T cells was measured at day 4. (c) A similar experiment using CD8 positive T cells. The values represent the mean ± s.d. of 12 parallel wells [please note that in the experiments shown in (b) and (c) five times more cells were used]. (d) CD8 positive T cells (derived from the lavage of patient 11) were cultivated in the presence of autologous PMN and SEA (10 ng/ml). After 24 h, synthesis of IFN-γ was measured by cytofluorometry of permeabilized cells (line IgG isotype control; filled peak anti-IFN-γ).