Effect of MALP-2, a lipopeptide from Mycoplasma fermentans, on bone resorption in vitro

Abstract

Mycoplasmas may be associated with rheumatoid arthritis in various animal hosts. In humans, mycoplasma arthritis has been recorded in association with hypogammaglobulinemia. Mycoplasma fermentans is one mycoplasma species considered to be involved in causing arthritis. To clarify which mycoplasmal compounds contribute to the inflammatory, bone-destructive processes in arthritis, we used a well-defined lipopeptide, 2-kDa macrophage-activating lipopeptide (MALP-2) from M. fermentans, as an example of a class of macrophage-activating compounds ubiquitous in mycoplasmas, to study its effects on bone resorption. MALP-2 stimulated osteoclast-mediated bone resorption in murine calvaria cultures, with a maximal effect at around 2 nM. Anti-inflammatory drugs inhibited MALP-2-mediated bone resorption by about 30%. This finding suggests that MALP-2 stimulates bone resorption partially by stimulating the formation of prostaglandins. Since interleukin-6 (IL-6) stimulates bone resorption, we investigated IL-6 production in cultured calvaria. MALP-2 stimulated the liberation of IL-6, while no tumor necrosis factor was detectable. Additionally, MALP-2 stimulated low levels of NO in calvaria cultures, an effect which was strongly increased in the presence of gamma interferon, causing an inhibition of bone resorption. MALP-2 stimulated the bone-resorbing activity of osteoclasts isolated from long bones of newborn rats and cultured on dentine slices without affecting their number. In bone marrow cultures, MALP-2 inhibited the formation of osteoclasts. It appears that MALP-2 has two opposing effects: it increases the bone resorption in bone tissue by stimulation of mature osteoclasts but inhibits the formation of new ones.

FIG. 1

Effect of MALP-2 on calcium release by murine calvaria. Pairs of calvarium halves were preincubated for 24 h in α-MEM containing 0.1% bovine serum albumin and then cultured for 72 h in the presence or absence of increasing amounts of MALP-2. Bone resorption is presented as the difference in the percentage of calcium release between the treated half (T) and the control half (C). Values are means ± SEM of 15 calvarium pairs. ∗, significantly different from control (no MALP-2) value; P < 0.001.

FIG. 2

Effects of inhibitors of prostaglandin synthesis on MALP-2-mediated bone resorption. Both halves of calvarium pairs were preincubated as described for Fig. 1 and then treated with either MALP-2 or vehicle without MALP-2 (no MALP-2). Indomethacin (open bars), hydrocortisone (hatched bars), and flurbiprofen (solid bars) at a concentration of 10⁻⁶ M were added to one half of the calvarium pairs, and the bones were incubated for 72 h. Values are means ± SEM of 10 pairs (MALP-2) and 5 pairs (no MALP-2). ∗, ∗∗, and ∗∗∗, significantly different from control (no inhibitor of prostaglandin synthesis) value; P < 0.05, P < 0.01, and P < 0.001, respectively.

FIG. 3

Effect of IFN-γ on NO production and MALP-2-induced bone resorption in murine calvaria. After 24 h of preincubation, the calvarium pairs were cultured for 48 h in the presence of MALP-2. One half was (hatched bars) and the other half was not (open bars) treated with IFN-γ (200 U/ml). NO production (A) and bone resorption (B) are given as means ± SEM of five calvarium halves. SEMs smaller than the symbols are not drawn. ∗ and ∗∗, significantly different from value for untreated half; P < 0.05 and P < 0.001, respectively.

FIG. 4

Effects of inhibitors of NO production on bone resorption. Two groups of calvarium pairs were investigated after preincubation as for Fig. 3. In one group, the effect of IFN-γ on pairs treated with MALP-2 (open bars) and MALP-2 and plus IFN-γ (200 U/ml) (hatched bars) was investigated. In the second group, the effect of 0.1 mM LMMA on calvarium pairs treated with MALP-2 plus IFN-γ (200 U/ml) (hatched bars) and MALP-2, IFN-γ, and 100 μM LMMA (solid bars) was investigated. NO production (A) and bone resorption (B) are given as means ± SEM of five halves. SEMs smaller than the symbols are not drawn. ∗ and ∗∗, significantly different from value for MALP-2 alone; P < 0.05 and P < 0.01, respectively. # and ##, significantly different from value for MALP-2 and IFN-γ; P < 0.05 and P < 0.01, respectively.

FIG. 5

Effect of MALP-2 on the bone-resorbing activity of isolated osteoclasts. Rat osteoclasts adhering to dentine slices were incubated for 24 h in the presence of vehicle (0 nM MALP-2) and various concentrations of MALP-2. The number of TRAP-positive multinucleated cells (osteoclasts; open bars) and of pits (hatched bars) (A) and the ratio of pits to TRAP-positive mononuclear cells (B) are given as means ± SEM of n (given in parentheses) slices. ∗, significantly different from value for 0 nM MALP-2; P < 0.05.