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. 2005 Mar 18;64(10):1436–1442. doi: 10.1136/ard.2004.034132

Transdifferentiation of polymorphonuclear neutrophils to dendritic-like cells at the site of inflammation in rheumatoid arthritis: evidence for activation by T cells

C Iking-Konert 1, B Ostendorf 1, O Sander 1, M Jost 1, C Wagner 1, L Joosten 1, M Schneider 1, G Hansch 1
PMCID: PMC1755243  PMID: 15778239

Abstract

Objectives: To investigate infiltrated cells in the synovial fluid (SF) of inflamed joints of patients with rheumatoid arthritis (RA), with special reference to polymorphonuclear neutrophils (PMN) and their interaction with T cells.

Methods: Expression on PMN of activation associated receptors CD14, CD64, CD83, and major histocompatibility complex (MHC) class II was examined in the SF of 15 patients with RA, as were the infiltrated T cells. SF cytokines were determined by enzyme linked immunosorbent assay (ELISA). To mimic the in vivo situation, co-culture experiments were carried out using PMN and T cells of healthy donors.

Results: The SF contained activated T lymphocytes and abundant PMN. SF PMN expression of CD14 and CD64 was enhanced compared with peripheral blood. Of special interest was the observation that only the SF PMN expressed MHC class II antigens and CD83. Exposure to SF, which contained considerable amounts of cytokines (for example, interferon γ (IFNγ), tumour necrosis factor α, and interleukin 2), induced a similar receptor pattern on blood derived PMN of healthy donors. Furthermore, PMN acquired MHC class II and CD83 within 24 to 48 hours, when co-cultured with autologous T cells or T cell lines. This effect was also achieved by T cell supernatants, was dependent on protein synthesis, and could be inhibited by antibodies against IFNγ.

Conclusions: SF PMN from patients with RA undergo major alterations, including transdifferentiation to cells with dendritic-like characteristics, probably induced by T cell derived cytokines. Because MHC class II positive PMN are known to activate T cells, the mutual activation of PMN and T cells might contribute to the perpetuation of the local inflammatory process, and eventually to the destructive process in RA.

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Figure 1.

Figure 1

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 Expression of surface receptors on leucocytes from synovial fluid (left panels) or peripheral blood of the same patient (right panels). (A) By forward-side scatter three major cell populations could be identified in the synovial fluid: R2 are PMN; R3, monocytes; R4, lymphocytes. Distribution of cells in the blood was similar. (B) Left panels: By double labelling PMN were identified by anti-CD66b FITC as a marker for PMN (in R7; right quadrant), and either IgG PE (as negative control), or PE conjugated antibodies to CD14, CD64, CD83, and MHC class II. Monocytes express similar antigens as PMN (except CD66b); they appear in R6 (middle quadrant) as a distinct cell population, constitutively expressing MHC class II and CD14. The lymphocytes (in R5; left quadrant) are negative for CD14, CD64, CD66b, CD83, but express MHC class II antigens. In whole blood (right panels), PMN are identified by anti-CD66b and are located in the lower and upper right quadrants. Monocytes and lymphocytes are co-located in the lower and upper left quadrants, both negative for CD66b.

Figure 2.

Figure 2

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 Expression of surface receptors on leucocytes from SF and peripheral blood of all 15 patients and of 20 NHD. The data are summarised as the percentage of positive PMN or as mean fluorescence intensity as detected by cytofluorometry by box and whiskers plots. The box contains 50% of the values, the square indicates the mean values, the horizontal bar the median. Differences between the groups were calculated using analysis of variance; the mean values were considered different when p was <0.05 (ND indicates no difference between the groups). There was no difference in the receptor expression pattern on SF PMN between patients with early and longstanding RA.

Figure 3.

Figure 3

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 Induction of CD83 and MHC class II expression on SF derived PMN derived cultured ex vivo. (A) Expression of CD83 and MHC class II was measured either directly after recovery of the PMN from the SF (line), or after culture for 16 hours at 37°C (filled peaks). (B) In parallel, activation associated antigens (CD69 and CD25) were determined on T cells after 16 hours (one of three experiments with cells of three patients is shown).

Figure 4.

Figure 4

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 Cytokines in the SF of all 15 patients: the cytokines were quantified by ELISA and expressed as pg/ml. Data of all patients are summarised as box and whiskers plots. The box contains 50% of the values, the square indicates the mean values, the horizontal bar the median (note, there is a logarithmic y axis). The dots at the bottom symbolise patients who were negative for the respective cytokines. Data from previous studies (unpublished observation; not shown) suggested that pretreatment of the SF with hyaluronidase and filtration of the samples to remove rheumatoid factors is not necessary.

Figure 5.

Figure 5

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 Activation of PMN in whole blood of NHD by SF of patients with RA. The left panel shows that culture for 48 hours activated PMN as seen by the expression of CD83 (line: cells cultured with PBS alone; filled peak: cultured with 50% SF; PMN were identified by anti-CD66b and gated accordingly). The right panel summarises the results of three independent experiments carried out with the blood of three healthy donors (NHD 1–3) and the SF of two patients with RA (SF-P1 and SF-P2); the CD83 expression after 48 hours stimulation with SF compared with PBS alone is shown.

Figure 6.

Figure 6

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 (A) Induction of MHC class II (HLA DP-DQ-DR) expression on PMN of a healthy donor after stimulation with 20% vol/vol supernatant (SN) of the T cell clone A37/2 for 48 hours (line: culture medium alone, filled peak: 20% SN added). (B) The acquisition of MHC class II was dependent on the SN concentration (10–50% vol/vol) (note: the experiment was performed with cells of a different donor). (C) The effect of the SN could be inhibited by antibodies to IFNγ and to a lesser extent by anti-GM-CSF (concentration 2 µg/ml each; conditions: 30% SN of the clone A37/2 for 24 hours; mouse IgG as isotype control did not inhibit the induction of MHC class II).

Figure 7.

Figure 7

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 (A) Induction of MHC class II (HLA DP-DQ-DR) expression on PMN of a healthy donor after co-culture with T cells (clone A37/2; 1x106 cells each/ml for 24 hours). Line: IgG PE isotype control; filled peak: MHC class II PE (HLA DP-DQ-DR). (B) The preincubation of T cells with cycloheximide (0.1 µg/ml for 2 hours) led to a decrease in MHC class II positive PMN (–39.6%) (filled peak: MHC class II expression on PMN after culture with untreated T cells; line: MHC class II expression after culture with cycloheximide treated T cells). (C) Number of MHC class II positive PMN with increasing numbers of T cells (clone A37/2) after 24 hours co-culture (1x106 PMN with 6.25x104–1x106 T cells/ml).

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