Anticardiolipin in porphyromonas gingivalis antisera causes fetal loss in mice.

ß2-glycoprotein I (ß2GPI)-dependent anticardiolipin autoantibodies (aCl) are associated with thrombosis and fetal loss. Some microbial pathogens can induce pathogenic antibodies cross-reactive with ß2GPI. Sera from a significant percentage of periodontitis patients contain aCl, and some periodontal pathogens contain antigens with peptide sequences having homology to ß2GPI. We hypothesized that antibodies raised against P. gingivalis (aPg) contain pathogenic aCl that induce fetal resorption. We immunized mice with ß2GPI, P. gingivalis W83, or an arg-gingipain-defective mutant of P. gingivalis (HF18). IgG fractions of aPg were immunoabsorbed to remove aCl-like antibodies (abs-aPg). IgG fractions were administered intravenously into tail veins of mated BALB/c females at day 0 of pregnancy. At day 15, the proportions of fetal resorptions were evaluated. The prevalence of fetal loss was significantly greater in the aPg group than in the control IgG group (21.2% vs. 5.3%, p = .001), and greater in the aPg group than in the abs-aPg group (21.2% vs. 12%, p < .05). There were no fetal resorptions observed in the aPgHF18 group (p = .0005 compared with aPg, p = .17 compared with control). aPg antibody contains activity consistent with pathogenic aCl, and the antigen inducing the antibodies that cause increased fetal loss may be on the arg-gingipain protease of P. gingivalis.

Anti-phosphorylcholine-opsonized low-density lipoprotein promotes rapid production of proinflammatory cytokines by dendritic cells and natural killer cells.

BACKGROUND AND OBJECTIVE: Epidemiological and animal studies suggest that periodontal infections increase atherosclerosis risk. Periodontitis patients have elevated levels of anti-phosphorylcholine (anti-PC) reactive not only with numerous periodontal organisms but also with minimally modified low-density lipoprotein (mmLDL). Dendritic cells (DCs) reside in arterial walls and accumulate in atherosclerotic lesions. The ability of anti-PC to bind mmLDL prompted the hypothesis that opsonized mmLDL would stimulate DCs and enhance the production of proinflammatory cytokines that promote atherogenic plaque development. MATERIAL AND METHODS: Monocyte-derived DCs (mDCs) were generated using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, then stimulated with mmLDL or with anti-PC-opsonized mmLDL. The anti-PC effect was determined using flow cytometry, cofocal microscopy and cytokine assays. The production of CD83, IL-12p35 mRNA, IL-12p40 mRNA, IL-12p70 and IL-10 by DCs was monitored. RESULTS: Dendritic cells stimulated with mmLDL expressed little CD83 and produced little IL-12p70. However, anti-PC-opsonized mmLDL enhanced DC maturation, as indicated by upregulated CD83 and rapid (≤ 48 h) production of IL-12p70 if a source of interferon-γ (IFN-γ) was available. In leukocyte cultures, natural killer (NK) cells rapidly produced IFN-γ (≤ 48 h) when interacting with IL-12-producing DCs activated by anti-PC-opsonized mmLDL. Moreover, IFN-γ promoted DC IL-12 responses that were further augmented when mmLDL was opsonized with anti-PC. CONCLUSION: Minimally modified LDL-stimulated DCs and NK cells were mutually stimulatory, with DC IL-12p70 needed by NK cells and with NK cell IFN-γ needed by DCs. Moreover, production of these proinflammatory cytokines was markedly enhanced when LDL was opsonized by anti-PC. In short, the data suggest that the elevated anti-PC levels in periodontitis patients could promote a mechanism that facilitates atherosclerosis.

Dendritic-NK cell interactions in P. gingivalis-specific responses.

Patients with localized aggressive periodontitis have type-1 cytokines in gingival crevicular fluid and high titers of IFN-gamma-dependent IgG2 reactive with P. gingivalis in gingival crevicular fluid and serum. Localized aggressive periodontitis monocytes spontaneously differentiate into dendritic cells that can stimulate IFN-gamma production by NK cells. These relationships prompted the hypothesis that P. gingivalis-dendritic cell-NK cell interactions might promote type-1 cytokine responses. Although P. gingivalis is not a potent inducer of Th1 responses, it stimulated strong IL-12 responses by monocyte-derived dendritic cells in the presence of IFN-gamma, and IFN-gamma was produced by NK cells within 24 hrs in the presence of dendritic cells. Anti-P. gingivalis IgG2 responses were enhanced by dendritic cells, and removal of NK cells reduced IFN-gamma- and P. gingivalis-specific IgG2. Thus, P. gingivalis-dendritic cell-NK cell interactions apparently resulted in reciprocal stimulation and increased type-1 cytokine production by both dendritic cells and NK cells, and increased P. gingivalis-specific IgG2.

Effects of thrombospondin purified by heparin affinity or ion-exchange chromatography on the alternative complement pathway.

Thrombospondin (TSP), a platelet glycoprotein, was purified from platelet supernatants applied sequentially to Sepharose 4B and heparin-agarose affinity columns. This TSP inhibited alternative pathway activity in serum as assessed by lysis of rabbit erythrocytes and contained bound heparin, a substance which is known to inhibit the alternative pathway. TSP purified by anion exchange chromatography did not contain heparin and was not inhibitory. Chromatography of this TSP over a heparin-agarose affinity column resulted in TSP which contained heparin and inhibited the alternative pathway. Purification of TSP by the standard technique of heparin affinity chromatography results in preparations which are contaminated with heparin.

Covalent linkage of C3 to properdin during complement activation.

Activation of the alternative complement pathway of serum produces complexes of properdin (P) and C3 as measured in a double antibody enzyme-linked immunosorbent assay. When purified from serum, these complexes decrease factor B hemolytic activity in serum and do not restore the alternative pathway hemolytic activity of serum deficient in P. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of activated serum containing biotinylated P, followed by blotting to nitrocellulose and development with streptavidin-alkaline phosphatase revealed a band at 53 kDa for monomeric P and an additional band at 160 kDa. P samples eluted from zymosan and purified from activated serum revealed a band at 116 kDa for C3 alpha and 74 kDa for C3 beta, and an additional band at 160 kDa when analyzed by SDS-PAGE, Western blotting and development with antibody to C3. The appearance of a 160 kDa band containing P and C3 indicates that these proteins are contained in a complex formed during activation of the alternative pathway. Activation of a purified reagent mixture containing factors B, D, and H, and 125I-labeled P or 125I-labeled C3, followed by SDS-PAGE and autoradiography confirmed the presence of a 160-kDa band which disappeared following hydroxylamine treatment of the sample. These data are consistent with a covalent linkage of C3 to P via the C3 alpha chain, producing the 160-kDa complex.

Association of activated properdin with complexes of properdin with C3.

An ELISA using antibody to properdin (P), followed by antibody to C3 to detect complexes of P with C3 (P-C3), detected low levels of P-C3 complexes in human serum and plasma samples. Incubating serum for 1 h at 37 degrees C increased the amount of P-C3 and diminished factor B hemolytic activity without altering total alternative pathway activity or C3 activity in serum. When P and C3 in incubated serum were analyzed by size exclusion HPLC, complexes of P-C3 were detected at retention times corresponding to molecular mass measuring in excess of 2 x 10(6) Da. Activation of serum with zymosan or cobra venom factor greatly increased the level of P-C3 and decreased alternative pathway hemolytic activity. Chromatography of proteins eluted from serum-treated zymosan detected a peak of P at 9.7 x 10(5) Da and a peak of P-C3 at 1.5 x 10(6) Da. Functional assays for activated properdin also revealed a peak of activity at 1.5 x 10(6) Da, congruent with the peak of P-C3. Native properdin was detected at 3.9 x 10(5) Da. When native properdin was added to properdin-depleted serum and incubated for 1 h at 37 degrees C, activated properdin was detected at the same position in the chromatograph as were P-C3 complexes. We conclude that incubation of serum at 37 degrees C produces complexes of P with C3, that exposure of serum to alternative pathway activators increases the amount of P-C3, and that generation of P-C3 complexes is associated with the presence of activated P.

Inherited deficiency of properdin and C2 in a patient with recurrent bacteremia.

A nine-year-old white boy with recurrent pneumococcal bacteremia is described. His serum had no hemolytic activity in either the classic or alternative complement pathways. Absence of classic pathway activity was secondary to a homozygous deficiency of C2. The parents had half-normal levels of C2, compatible with an autosomal recessive mode of inheritance. Measurement of serum properdin levels by radial immunodiffusion and enzyme-linked immunoabsorbent assay revealed a profound deficiency in the patient, normal levels in the father, and half-normal levels in the mother, suggesting X-linked inheritance of the deficiency. Addition of purified properdin to the patient's serum fully reconstituted the alternative pathway function. This patient's unique combination of inherited deficiencies of properdin and C2 is a likely explanation for his susceptibility to bacterial infection.