In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.
OBJECTIVE
The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs).


METHODS
Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement.


RESULTS
The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1β, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18).


CONCLUSIONS
Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.
Section for Periodontology, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.|Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
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Damgaard
Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark.
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J
Reinholdt
Unit for Thrombosis Research, Institute of Public Health, University of Southern Denmark, Esbjerg, Denmark.
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Y
Palarasah
Section for Periodontology, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.|Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
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Enevold
Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
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C
Nielsen
Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
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MK
Brimnes
Section for Periodontology, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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Holmstrup
Section for Periodontology, Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.|Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
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Nielsen
D000328:Adult; D000368:Aged; D016976:Aggregatibacter actinomycetemcomitans / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002448:Cell Adhesion; D003167:Complement Activation; D016207:Cytokines / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism / Q000382:microbiology*; D010861:Fimbriae, Bacterial / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D016753:Interleukin-10 / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000382:microbiology*; D008875:Middle Aged; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D055815:Young Adult
D001426:Bacterial Proteins; D016207:Cytokines; D006460:Hemolysin Proteins; D053583:Interleukin-1beta; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; C102677:lktA protein, bacteria; D016753:Interleukin-10

Aggregatibacter actinomycetemcomitans
; complement; cytokines; monocytes; red blood cells