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International Immunology Advance Access originally published online on January 13, 2006
International Immunology 2006 18(3):459-464; doi:10.1093/intimm/dxh386
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Pasteurella multocida toxin (PMT) activates RhoGTPases, induces actin polymerization and inhibits migration of human dendritic cells, but does not influence macropinocytosis

Dagmar Blöcker1,*, Luciana Berod2,*, Joachim W. Fluhr2, Joachim Orth1, Marco Idzko3, Klaus Aktories1 and Johannes Norgauer2

1 Department of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, D-79104 Freiburg, Germany
2 Department of Dermatology, Friedrich Schiller University, Erfurter Strasse 35, D-07740 Jena, Germany
3 Department of Pneumology, University of Freiburg, D-79104 Freiburg, Germany

Correspondence to: J. Norgauer; E-mail: johannes.norgauer{at}med.uni-jena.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) are considered as one of the principal initiators of immune responses. In their immature state, they migrate into peripheral tissue in order to uptake antigen and to patrol for danger signals. Upon maturation, they acquire the ability to migrate to the lymph nodes and present the captured antigens to T cells in order to direct the development of specific immune responses. There is evidence that microbial compounds interfere with proper functions of DCs in order to block innate and specific immunity. Here we characterized the influence of Pasteurella multocida toxin (PMT) on monocyte-derived DCs. Using pull-down assays with recombinant rhotekin or p21-activated kinase, we demonstrated the activation of RhoGTPases by PMT in DCs. Moreover, PMT induced changes in DC morphology and actin polymerization, impaired chemotaxin-induced actin re-organization and inhibited their migration response. However, macropinocytosis was not influenced by PMT. In summary, these data indicate that PMT inhibits proper function of the motility machinery in DCs, which might limit the development of adaptive immune surveillance during infection with Pasteurella multocida.

Keywords: actin, dendritic cells, migration, Pasteurella multocida toxin


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Pasteurella multocida is a small gram-negative coccobacillus that often resides the gastrointestinal tract and nasopharynx of wild and domesticated animals including cats and dogs. Whereas it causes rarely disease in its animal hosts, in humans P. multocida causes cellulitis and localized superficial skin abscesses following an animal bite or scratch (1). The respiratory tract is the second most common site of infection for P. multocida in humans. Moreover, P. multocida is able to cause osteomyelitis, intra-abdominal infections, septic arthritis, sepsis and meningitis (1–3).

One of the major virulence factors of P. multocida is a toxin with a molecular mass of 146 kDa, called Pasteurella multocida toxin (PMT) (4, 5). This toxin is an extremely potent mitogen for Swiss 3T3 cells and fibroblasts (4, 6). Previous studies have shown that the cellular effects of PMT are mediated at least by two different types of GTPases (4, 7, 8). PMT stimulates the Gq/11 GTPase signalling pathway and subsequently activates phospholipase (PL) Cß1 (9). Indeed, PMT-treated Xenopus oocytes have increased inositol 1,4,5-trisphosphate and intracellular free Ca2+ levels (9). Moreover, PMT induces tyrosine phosphorylation of focal adhesion kinase and paxillin (4, 7) and stimulates actin stress fiber formation (6, 10). The latter effects are suggested to be mediated by the activation of small RhoGTPases (6, 7, 10). Interestingly, the activation of Rho is independent of Gq proteins (11). However, the precise target and molecular mechanism of PMT is not yet known.

Dendritic cells (DCs) are antigen-presenting cells specialized in initiating adoptive immune responses (12–14). They are originated from haemopoietic stem cells in the bone marrow and migrate in their immature form into peripheral sites in order to uptake antigen and to patrol for danger signals (12–14). Under steady-state conditions, there is a very limited efflux of DCs from peripheral tissue to the lymph nodes (15, 16). In contrast, when tissues are perturbed by inflammatory changes, DCs undergo a maturation process (15, 16), which is associated with the lose of the phagocytotic capacity, enhanced migration to the T cell areas of secondary lymphoid organs, increased membrane expression of MHC and co-stimulatory molecules as well as the acquisition of potent T cell-stimulating functions (12–14). In secondary lymphoid organs, mature DCs present the captured antigens to T cells and direct the development of specific T- and B-cell immune responses (12).

Currently, the knowledge about the influence of PMT on immune cells is limited. In vivo experiments suggested that the crude extract of PMT is poorly immunogenic and does not initiate a protective and specific immune response (17, 18). It has been recently reported that PMT induces maturation of DCs through activation of PLC and subsequent mobilization of intracellular calcium (19). In vitro, this activation of DCs is accompanied by enhanced stimulation of naive alloreactive T cells, meanwhile in vivo PMT antibody production is suppressed (19).

In order to better understand the interaction of P. multocida with the immune system, we have characterized the influence of PMT on DCs. Here we report that in DCs, PMT activates RhoGTPases, induces actin polymerization and inhibits migration, but does not influence macropinocytosis.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents
Recombinant human complement fragment 5a (C5a) was purchased from Sigma (Deisenhofen, Germany), RANTES (CCL5) and macrophage inflammatory protein-3ß (CCL19) from Peprotech (London, UK).

Preparation of PMT
PMT or the inactive PMT-C1165S were expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein and purified as described (7, 9, 20). Contaminating LPS was removed by polymyxin B treatment (21). The final concentration of LPS in the toxin preparations was below the detection limits of the limulus amebocytes test (21). The cytotoxic-necrotizing factor (CNF) 1 from E. coli was prepared as described (22).

Preparation of human DCs
PBMCs were prepared from buffy coats of healthy donors using Ficoll-Paque gradients (Amersham–Pharmacia Biotech AB, Uppsala, Sweden). After centrifugation, cells were separated with anti-CD14 mAb-coated MicroBeads using MACS single use separation columns from Miltenyi Biotec (Bergisch Gladbach, Germany). The CD14+ cells were re-suspended in RPMI 1640 medium containing 10% FCS 1% glutamine, 50 IU ml–1 penicillin, 50 µg ml–1 streptomycin (all from Life Technologies GmbH, Karlsruhe, Germany), 1000 U ml–1 IL-4 and 10 000 U ml–1 granulocyte macrophage colony-stimulating factor (Natutec, Frankfurt, Germany). After 5 days of culture, cells were >95% CD1a+ and CD14–. Further maturation was obtained by incubating DCs with 3 µg ml–1 LPS (Sigma, St Louis, MO, USA) for 24 h. Mature DCs expressed CD83 and up-regulated expression of HLA-DR, CD54, CD80 and CD86 molecules, as assessed by flow cytometry. All mAbs and the respective isotype-matched controls were from Coulter-Immunotech (Krefeld, Germany) (23).

Actin polymerization
The content of filamentous actin (f-actin) was analyzed by flow cytometry after fixing the cells and staining them with N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)-phallacidin (Becton Dickinson, Heidelberg, Germany). The relative f-actin content after toxin treatment or stimulation with chemotaxins is given as ratio of the mean channel number of the indicated sample with respect to the untreated control cells (24).

Migration assay
Experiments were performed in 48-well plates (Nucleopore, Tübingen, Germany). The upper and lower compartments were separated by a 5-µm pore polycarbonate membrane. DCs were added to the top chamber, while the bottom chamber contained medium (control) or the indicated chemoattractant. After 90 min, DCs present on the lower side of the membrane were fixed with methanol and stained with haematoxylin. Five randomly chosen microscopic fields were counted. The chemotactic index was calculated as the ratio between stimulated and control medium-treated cells (25).

Macropinocytosis assay
Immature DCs were washed, re-suspended in complete medium and pulsed with 1 mg ml–1 Texas red-conjugated BSA. Thereafter, DCs were incubated at 37 or 4°C for 1 h. Uptake was stopped by adding cold buffer containing 2% FCS and 0.01% NaN3. Cells were then washed four times and analyzed by flow cytometry. Surface binding values obtained by incubating cells at 4°C were subtracted from values measured at 37°C (26).

Rhotekin and p21-activated kinase pull down assay
The binding region encoding the N-terminal 90 amino acids of rhotekin or the Cdc42/Rac-interactive binding domain encoding amino acids 56–272 of p21-activated kinase (PAK) were expressed as GST-fusion protein in E. coli BL21 and purified by affinity chromatography with glutathione–Sepharose (Amersham–Pharmacia Biotech, Freiburg, Germany). Loaded beads were incubated with the cytosolic fractions of DCs for 1 h at 4°C by head-over-head rotation. After washing steps, the samples were boiled and separated on SDS-PAGE. RhoA and Rac were analyzed by immunoblotting (7, 27).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
PMT stimulates RhoGTPases and morphogical changes in DCs
PMT has been shown to induce actin re-organization through activation of the RhoGTPase. To test whether this pathway was also activated in DCs, we have analyzed Rho activation after PMT treatment using pull-down assays with GST–rhotekin, which specially binds the active GTP-bound forms of Rho. As shown in Figure 1A, PMT and CNF1 from E. coli stimulated binding of activated Rho to GST–rhotekin. A typical western plot of the input into the GST–rhotekin assay confirmed equal loading of Rho proteins in the analyzed samples. In contrast, PAK pull down, an assay for Rac activation, showed activation with CNF1, but not with PMT (Fig. 1B). Moreover, RhoGTPases have been involved in the regulation of various morphological changes in cells. Figure 2 shows the typical characteristic morphology with irregular shape and cytoplasmic veils of immature DCs. After PMT treatment, DCs experimented a remarkable morphological change, leading to spread cells with long dendritic needle-like extensions.


Figure 1
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  		Fig. 1. PMT activates RhoGTPases. Immature DCs were treated without or with 500 ng ml–1 PMT, the inactive PMT-C1165S form or 500 ng ml–1 CNF for 3 h, respectively. Thereafter, rhotekin (Fig. 1A) or Cdc42/Rac-interactive PAK binding domain (Fig. 1B) pull down experiments were performed as described in Methods. Representative western blots of Rho and Rac activation, respectively (top panel), compare to the input of the assays (bottom panel) are shown. Experiment was repeated three times with similar results.

 

Figure 2
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  		Fig. 2. Effect of PMT on DC morphology. Immature DCs were treated without or with 500 ng ml–1 PMT for 3 h. The morphologic changes were analyzed by invert microscopy. The results are representative of three independent experiments.

 
PMT induces actin polymerization in both mature and immature DCs
Previous reports indicate that RhoGTPases are involved in the regulation of actin structures in different cells (10). Therefore, the effects of PMT and the chemotaxin C5a on f-actin were analyzed by flow cytometry (Fig. 3A). An increase of the actin content was induced by optimal concentrations (10 µg ml–1) of C5a in immature DCs. The response was transient and the f-actin content was recovered to initial values after 600 s. Incubation of DCs with PMT for 2 h increased the basal level of the actin content in immature DCs by ~25%. However, C5a was still able to enhance the f-actin content in a time-dependent manner, despite the relative amplitude of f-actin changes was decreased. After 4 h incubation with PMT, 1.75-fold higher basal levels of f-actin content, in comparison to control DCs, were detected and the relative effect of C5a was further reduced. After 8 h incubation with PMT, 2.25-fold higher levels of f-actin were seen, and C5a was not anymore able to influence the f-actin content, probably because maximal actin levels are already induced by PMT. Indeed, similar effects of PMT in immature DCs were also observed after stimulation with other chemotaxins such as CCL5 (Fig. 3B).


Figure 3
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  		Fig. 3. PMT induces actin polymerization and impairs actin re-organization in DCs. (A) Immature DCs were treated without or with 100 ng ml–1 PMT for indicated time. Thereafter, cells were stimulated with 10 µg ml–1 C5a for the indicated times. (B) Immature and LPS-matured DCs were treated without or with 100 ng ml–1 PMT for 4 h. Thereafter, immature or LPS-matured cells were stimulated for 25 s with 10 µg ml–1 C5a, 10 nM CCL5 or 10 nM CCL19, respectively. (C) Immature DCs were treated without or with 100 ng ml–1 PMT or PMT-C1165S for 4 h. Thereafter, cells were stimulated for 25 s with 10 µg ml–1 C5a. The relative f-actin content was analyzed by flow cytometry. Data are expressed as means ± SD (n = 3).

 
To test whether this effect of the PMT was maturation-stage specific, we next checked the effect over mature DCs, which express the CC-chemokine receptor 7, necessary for the trafficking from peripheral sites to secondary lymphoid organs. In LPS-matured DCs, PMT induced also f-actin formation and impaired the transient CCL19-induced actin response (Fig. 3B). Thereafter, we analyzed the effect of the inactive mutant PMT-C1165S on actin in DCs. In contrast to the Rho-activating PMT, this inactive mutant was unable to provoke actin polymerization (Fig. 3C).

PMT inhibits migration, but does not influence macropinocytosis
Actin re-organization is a prerequisite for migration of leukocytes (24, 28). Migration of DCs is regulated by chemotaxins such as C5a. Thus, considering the similar ability of C5a and PMT in inducing actin polymerization, we analyzed whether PMT had chemotactic activity for DCs. Performed Boyden chamber experiments provided no evidence that PMT acts as a chemotaxin for DCs (data not shown). However, pre-treatment of immature DCs with PMT totally inhibited C5a-induced chemotaxis (Fig. 4A). Similar results were seen in experiments using CCL5- and CCL19-stimulated immature and mature DCs, respectively (Fig. 4B).


Figure 4
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  		Fig. 4. PMT-blocked chemokine induced migration of DCs. (A) Immature DCs were treated without or with 100 ngml–1 PMT for 2 or 4 h. Thereafter, cells were exposed to 10 µg ml–1 C5a at 37°C for 90 min in a Boyden chamber. (B) Immature and LPS-matured DCs were treated without or with 100 ng ml–1 PMT for 4 h. Thereafter, cells were exposed to 10 µg ml–1 C5a, 10 nM CCL5 or 10 nM CCL19, respectively. The chemotactic index was calculated as reported in Methods. Data are means ± SD (n = 6).

 
Experiments with actin-disrupting agents indicated an essential role of the actin network on phagocytotic processes (29). However, flow cytometric experiments revealed that PMT did not significantly influence the macropinocytotic activity of immature DCs (Fig. 5).


Figure 5
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  		Fig. 5. PMT does not influence macropinocytosis in immature DCs. Immature DCs were treated without or with 100 ng ml–1 PMT for the indicated time. Subsequently, macropinocytosis was analyzed by flow cytometry, as indicated in Methods. Data are means ± SD (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
DCs are a heterogeneous group of migratory bone marrow-derived cells, specialized for the uptake, transport, processing and presentation of antigens to T cells. Under physiological conditions, DCs remain in an immature state, acting as sentinels in peripheral tissues (12–14). Any encounter with microbial products or tissue damage initiates the migration of DCs to the lymph nodes and a maturation process which is accompanied by the up-regulation of the co-stimulatory molecules required for the effective interaction with T cells (15–16).

Pasteurella multocida is a small gram-negative coccobacillus, which causes cellulitis, localized superficial skin abscesses, pneumonia, tracheobronchitis, sinusitis and pharyngitis in humans (1–3). A synthesized toxin, PMT, is one of the major virulence factors of this bacteria (1–3). Recently, it has been reported that PMT stimulates maturation of DCs through activation of PLC and subsequent mobilization of intracellular calcium (19). According to these data, we could also show that PMT induces actin polymerization and influences the characteristic morphology of immature DCs, leading to a mature typical morphology with long spindle-like dendritic processes. Indeed, a crucial role of RhoA in the regulation of the actin network and the morphological features is meanwhile well documented (6, 7, 10). In well accordance to this concept, here we show that PMT stimulates RhoA in DCs, but does not influence Rac activation (6, 7), indicating that RhoA might be responsible for these morphological changes.

Moreover, we found that PMT treatment impaired the reversible chemotaxin-induced actin polymerization. Experiments with actin-modifying toxins such as Clostridium botulinum C2 toxin or cytochalasins, indicated that actin polymerization is a prerequisite for phagocytosis and the cell motility response (24, 28, 29). Since PMT blocks chemotaxin-induced migration of DCs, one can assume that reversible actin cytoskeletal rearrangements are required for migration. In contrast to the migration response, flow cytometric studies reported here implicate that only an intact actin scaffold and not the reversibility of the actin dynamics is a prerequisite for phagocytotic activities such as macropinocytosis. Further studies with the actin-inducing PMT toxin might help in future to distinguish distinct requirements of actin dynamics in cell reponses.

Discordant effects of PMT on immune reaction in vitro and in vivo have been reported (17, 18). While in vitro, PMT-matured DCs enhanced stimulation of naive alloreactive T cells, in vivo they are able to suppress antibody production (19). Taking into consideration our data regarding the motility of DCs, this low immunological response in vivo might be explainable. Usually DCs seed and patrol peripheral tissues to collect antigens. After contact with SOS signals such as inflammatory or bacterial-derived molecules, DCs undergo maturation and increase their traffic to regional lymph nodes. In secondary lymphoid organs, mature DCs present the captured antigens to T helper cells and direct the development of specific T- and B-cell-dependent immune responses. Our present studies indicated that PMT does not influence the uptake of molecules. Moreover, T cell proliferation studies with pre-treated DCs provided no evidence that PMT inhibits activation of naive T cells (19). Therefore, inhibition of the trafficking response of DCs by PMT might abolish proper presentation of the antigens in secondary lymphoid organs blocking development of adoptive immune responses. Therefore, PMT by interfering with the motility machinery of DCs might program an elegant escape mechanism from adoptive immune surveillance during infection with P. multocida.

In summary, we have shown here that PMT activates RhoGTPases, induces morphological shape changes, provokes actin polymerization, impairs chemotaxin-induced actin re-organization and inhibits the migration response of DCs. Therefore, it is suggestive that PMT might impair the development of immune response by inference with the motility machinery of DCs.


   Acknowledgements
 
This work was supported by a grant of DFG (No. 266/3-1).


   Abbreviations
 
C5a   complement fragment C5a
CNF   cytotoxic-necrotizing factor
DC   dendritic cell
f-actin   filamentous actin
GST   glutathione-S-transferase
PAK   p21-activated kinase
PL   phospholipase
PMT   Pasteurella multocida toxin

   Notes
 
* These authors contributed equally to this manuscript. Back

Transmitting editor: M. Reth

Received 8 August 2005, accepted 15 December 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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