periodontal diseases are infectious diseases of humans and other mammals that result in damage to the soft tissue and periodontal ligaments of the gingiva , resorption of alveolar bone from the jaw , and , ultimately , loss of teeth .
it is becoming increasingly apparent that there is extensive interaction and cross - talk between the immune system and bone remodeling .
this new concept has wide - ranging implications for how we think about the causes and regulation of bone resorption and has engendered a new field called osteoimmunology [ 1 , 2 ] .
bone is constantly remodeling , with osteoblasts building bone and osteoclasts breaking it down . in a state of health , these two processes are in balance and the density of bone remains stable .
this balance is disrupted in various diseases , including the osteoclastic resorptive disease , chronic periodontitis . in this periodontal disease the progressive loss of alveolar bone
is largely activated by immune responses to a small subset of the more than 600 species of bacteria that colonize the oral cavity . in a murine model
that we have developed previously , we have shown that alveolar bone loss from the jaw can be triggered by oral infection with one of these 600 oral bacterial species , porphyromonas gingivalis , a periodontopathic gram - negative anaerobe from humans [ 3 , 4 ] .
we , and others , have shown that in response to oral infection b lymphocytes and cd4 t lymphocytes of the immune system trigger bone resorption [ 1 , 35 ] . despite the prevalence of periodontal disease ,
the pathways to bone loss are not fully understood . in the most well - characterized pathways , infection upregulates expression of ligand for receptor activator of nf-b ( rank - l ) on specific antigen - activated b and cd4 t lymphocytes .
these immune cells are then able to bind to rank on preosteoclasts , triggering bone resorption [ 47 ] .
we wished to explore whether there are other immune cell surface molecules that can induce bone loss . in this study
we have used mice deficient in their b lymphocyte expression of igd , a b cell surface immunoglobulin known to be involved in b cell / t cell cognate activation , but not known to be involved in bone remodeling . in immune normal mice and humans , expression of membrane bound igd along with igm indicates that a b cell has undergone affinity maturation and can begin to react with specific antigen and secrete soluble antibody to that antigen .
igh-5 mice have a targeted mutation that deletes expression of heavy chain-5 ( igh-5 ) , the delta - like heavy chain of igd immunoglobulin . mice deficient in igd display a delay in b cell affinity maturation .
in addition , b cell membrane igd is a ligand for igd receptors that are expressed on activated cd4 t cells .
binding of igd to igd receptors facilitates b cell antigen presentation to cd4 t cells by stabilizing t cell receptor binding to the antigen / mhc complex .
this cognate interaction increases both antibody responses by the b cells and antigen - specific clonal expansion of the t cells .
we therefore designed experiments using igh-5 mice to test the hypothesis that igd can contribute to alveolar bone loss in response to p. gingivalis infection .
specific pathogen - free balb / c - igh-5 mice that are igd deficient due to a specific deletion of the igh gene   and the genetically matched immune normal strain balb / cj were received from the jackson laboratory ( bar harbor , me , usa ) .
mice were kept in the animal colony at bates college under conditions described previously . the experimental protocol was reviewed and approved by the institutional animal care and use committee , bates college . 

p. gingivalis atcc 53977 ( a7a1 - 28 ) was maintained frozen in defibrinated sheep 's blood at   80c . for experiments , bacteria
were anaerobically grown under 5% co2 , 10% h2 and 85% n2 at 37c for 47 days on supplemented blood agar containing trypticase soy agar base with 0.1% yeast extract , hemin ( 5.0 g / ml ) , menadione ( 0.5 g / ml ) , and 5% defibrinated sheep 's blood .
bacteria were removed from the plates , gently suspended in phosphate - buffered saline ( pbs ) , and diluted to 10 colony forming units ( cfu ) per ml on the basis of optical density .
pilot experiments in which serial dilutions of p. gingivalis were plated for colony counts had shown that a 1:100 dilution of 10 cfu per ml of p. gingivalis has an optical density of 0.3 at 650 nm .
each experiment included ten age - matched female mice , 14 to 16 weeks old at the start of experiments .
mice were given sulfamethoxazole ( 0.87 mg / ml ) and trimethoprim ( 0.17 mg / ml ) ( sulfatrim , goldline laboratories , ft .
lauderdale , fl , usa ) , ad libitum in their drinking water for 10 days , followed by a 3-day antibiotic - free period .
mice were then infected by gavage needle and direct oral exposure three times at 2-day intervals with 10 cfu of live p. gingivalis in 100 l of pbs with 2% carboxymethylcellulose .
controls received the antibiotic pretreatment and three sham - infections with carboxymethylcellulose without p. gingivalis .
horizontal bone loss around the maxillary molars of defleshed skulls was assessed under a dissecting microscope ( 40 ) fitted with a video image marker measurement system ( model via 170 , boeckeler instruments , inc . ,
the distance from the cementoenamel junction ( cej ) , where the enamel of the tooth crown meets the cementum of the tooth root , to the alveolar bone crest of the jaw ( abc ) was measured at a total of 14 buccal sites per mouse ( for location of measurement sites see photographs in ) .
measurements were done in a blinded protocol by one investigator . as alveolar bone resorbs away ,
additional root cementum is exposed and the distance from the cej to the new location of the abc becomes larger . where the cej to abc is larger in infected mice than in sham - infected mice , bone loss has occurred .
the cej to abc distance is not zero ; consequently , the cej to abc distance at individual sites in infected mice was subtracted from the mean of the distance at that site in sham - infected mice , giving the mm of change in bone at that site . at the time of euthanasia , bacterial samples were collected from the oral cavity using one sterile paper - point per mouse placed along the cej for 5 seconds on the buccal surface of the three maxillary molars on the left and 5 seconds along the molars on the right .
sera were stored at   80c for later assessment of specific igm , igg , and iga antibody by enzyme - linked immunosorbent assay ( elisa ) , as described previously , in polystyrene plates ( costar , corning inc . ,
corning , ny , usa ) coated with formalin - killed whole p. gingivalis atcc 53977 .
the elisa titer was defined as the reciprocal of the highest serum dilution ( expressed in log2 ) that produced absorbance readings more than two standard deviations above background levels .
the p. gingivalis - specific titer of each infected animal was calculated by subtraction of the mean value from the sham - infected animals of that mouse strain .
fifty l of mouse spleen cells at a density of 2  10 cells / ml were washed in 4 ml of wash buffer ( 0.2 g kcl , 8.0 g nacl , 1.15 g na2hpo4 , 0.2 g kh2po4 and 0.2 g nan3 per liter at ph 7.2 ) and pelleted at 110    g for 10 minutes at 15c .
the pellets were resuspended in 1 ml of the same wash buffer and were blocked with 10 l of normal rat igg ( sigma chemical company , st .
cells were immunostained for 30 minutes at 7c with either 10 ul of prediluted hamster antimouse cd69 conjugated to fitc to show activation status , or phycoerythrin - conjugated hamster antimouse cd19 to mark b cells or hamster anti - mouse cd4 to mark cd4 t cells , and fitc- or phycoerythrin - labeled hamster igg as isotype controls ( the jackson laboratory , bar harbor , me , usa ) .
unadsorbed antibody was removed by adding wash buffer and centrifuging at 60    g for 10 minutes at 10c .
cells were resuspended in 0.5 ml flow cytometry buffer ( the wash buffer with 5 ml / l fetal bovine serum added ) .
five l of propidium iodide at 20 g / ml were added to determine cell viability .
data were acquired on a facsort ( becton - dickinson , san jose , ca , usa ) and were analyzed using cellquest .  
lymphocytes were gated on the basis of forward scatter and side scatter of incident light .
differences between the infected and sham groups and between infected mice of different strains were evaluated by two - tailed , unpaired t - tests ( excel ; microsoft , redmond , wa , usa ) .
the effects of oral infection with p. gingivalis on alveolar bone are shown in figure 1 .   in balb / cj mice with normal igd levels
the cej to abc distance was larger in infected mice than in sham - infected mice at each of the 14 measurement sites , indicating that the infected balb / cj mice had lost bone at every site ( figure 1(a ) ) .
in contrast , in igd deficient mice the cej to abc distances were the same in infected mice as in sham - infected mice , showing that without b cell igd mice did not lose bone at any site ( figure 1(b ) ) . summing the amount of bone change at all 14 sites additionally demonstrated that bone loss occurred in immune normal mice but not in igd deficient mice .
the summed change in infected b cell normal balb / cj mice was a mean of   0.302  0.035 mm of bone .
in contrast , the summed bone change in infected igd deficient mice was 0.040  0.024 mm ( p = 1.28  10 ) . to be sure that lack of bone loss could not be explained by a lack of infection , we reisolated p. gingivalis from the oral cavities by paper point .  
p. gingivalis was present in each of the infected mice of both strains and was not present in any of the uninfected mice ( data not shown ) .  
the p. gingivalis load was actually much higher ( a mean of 100  10 colonies per plate , n = 10 ) in the igd deficient mice than in the immune normal mice where the mean was only 2  1 colonies per plate ( data not shown ) .
the difference in bacterial load may reflect differences between the mouse strains in their production of p. gingivalis - specific serum antibody .  
after infection , both mouse strains produced a specific antibody response significantly higher ( p < .05 ) than the background levels in sham - infected animals ( data not shown ) ; however , the titers of each of the three antibody isotypes in the infected mice were lower in the igd deficient mice than in the immune normal mice ( p < .05 ) ( figure 2 ) . despite the differences in serum antibody titers ,
this was confirmed in our experiments by flow cytometry measurements on the day of euthanasia , 42 days postinfection by comparing the sham - infected mice of the two strains .
the mean percentage of b cells was higher in igd knockout mice than in normal mice , but the difference between the two mouse strains was not statistically significant ( figure 3(a ) ) . in neither mouse strain did p. gingivalis - infection change the percentage of total b cells compared with sham - infected mice ( figure 3(a ) ) .
although total b cell percentages were unchanged , a higher percentage of b cells were activated in infected balb / cj mice than in infected igd knockout mice ( p < .05 ; figure 3(b ) ) .
we also examined the cd4 t cell populations in the two mouse strains . in balb / cj mice
p. gingivalis - infection led to a significant increase in the mean percentage of total viable cd4 t cells compared with sham - infected balb / cj mice ( p < .05 ) .
in igd deficient mice , however , infection did not increase the cd4 t cell population ( figure 4(a ) ) .  
similarly , infection activated cd4 t cells in balb / cj mice , as shown by the increased percentage of cd69 cd4 t cells , but it did not activate t cells in igd deficient mice ( figure 4(b ) ) .
in this study , we show that mice with b cells lacking surface igd do not experience alveolar bone loss after oral infection with p. gingivalis ( figure 1 ) .   one possible explanation for the lack of bone loss could be that the mice were colonized by with a diminished load of p. gingivalis .
reisolation of the bacteria eliminated this possibility ; igd deficient mice actually harbored higher numbers of this bacterium than immune normal mice .
higher colonization may be due to the lower antibody titers found in the igd deficient ( figure 2 ) .
these reduced titers are in line with earlier studies showing that diminished antibody response is due to the delayed affinity maturation characteristic of this igd mutation .
previous studies in immune normal mice have demonstrated that antibody may decrease bacterial load but antibody is not protective against bone loss : lower antibody titers do not lead to more bone loss .
we can not entirely exclude another possibility ; that is , that the resistance to bone loss seen in the igd deficient mice was due to residual contamination with alleles from the 129 or c57bl/6 progenitor strains .  
the igh knockout was originally generated on the c57bl/6 genetic background by gene targeting with an embryonic stem cell line derived from murine strain 129 .  
balb / c - igh-5 mice were produced by backcross onto the balb / cj strain for seven generations .
strain balb / cj is genetically susceptible to bone loss after oral infection with p. gingivalis , while strains 129 and c57bl/6 show moderate resistance to bone loss in some experiments ; however , mice within each cohort do lose bone after infection . in seven backcross generations the progenitor genotypes
are predicted to be diluted 1 in 2 , leaving less than 0.8% of the progenitor alleles carried into the balb / cj genome and then only if they are closely linked to the target igh gene . in the experiments presented in this paper , no infected balb / c - igh-5 mouse showed an increase in the cej to abc distance ( our measure of bone loss ) that was beyond one standard error of the mean value of the shams ; that is , the distance from the cej to the abc in every infected mouse was within the range of variation of the sham mice .
we therefore think it highly unlikely that the absence of bone loss observed in the infected mice is the result of passenger 129 or c57bl/6 gene loci but is instead an intrinsic feature of the igh deficiency .
we propose that the resistance to bone loss is due to the inability of igd deficient b cells to act as costimulators of antigen - specific t cells .
t cells have igd receptors that are lectins and bind to o - glycans on b cell surface igd immunoglobulin .
these igd receptors are present on very low numbers of peripheral t cells but are moved to the surface membrane in response to activation by antigen .
the upregulated igd receptor on an antigen - specific t cell binds to igd on a b cell bearing the same antigen , stabilizing the t cell receptor / mhc ii connection between the two types of lymphocytes , facilitating activation of both .
inflammatory bone resorption triggered by aggregatibacter ( formerly actinobacillus ) actinomycetemcomitans requires activation of th1 cd4 t cells , and this activation requires costimulation of b7 on the t cell surface . blocking of b7 abrogated costimulation and inhibited bone resorption in a rat model .  
the present results suggest that b cell igd binding to cd4 t cell igd receptors is a pathway to costimulation of both b cells and cd4 t cells in p. gingivalis - induced disease .
our experiments show that oral infection of immune normal mice with p. gingivalis leads to both serum antibody response ( figure 2 , an indication of b cell activation ) and to higher numbers and activation of cd4 t cells ( figure 4 ) .
in contrast , igh-5 mice lacking igd showed signs of less activation of both b and t cells .
they produced lower antibody titers ( figure 2 ) , had fewer activated b cells ( figure 3 ) , and did not show an increase in t cell population or activation at 42 days after infection ( figure 4 ) .
the mechanism by which this cognate b and t cell activation stimulates bone loss is not known at this time .
it is not likely to be due to the effect of igd deletion on antibody secretion , as antibody is not protective against bone loss .
one well - studied immunological pathway to bone loss is upregulation of rank - l on activated lymphocytes .
both b cells and t cells express membrane bound and soluble rank - l after activation [ 16 , 17 ] , and bone loss can be triggered by the binding of this ligand to rank on preosteoclasts .
however , both cd4 and cd8 t cells can express rank - l after activation .
we have shown that cd4 t cells and their cytokines are involved in alveolar bone loss in mice , but that cd8 t cells are not .
others have also implicated cd4 cells [ 2 , 15 , 18 ] , suggesting that there may a pathway in addition to rank - l that is particular to cd4 but not cd8 t cells . although we have some preliminary data showing that the proportion of cd4 spleen cells expressing membrane surface rank - l was the same in infected immune normal mice that lost bone as it was in igd deficient mice that did not lose bone ( data not shown ) , we have not examined rank - l expression on t or b cells in the gingiva .
one pathway that could distinguish between cd4 and cd8 t cells is the pathway suggested by these experiments , that is , activation through the t cell igd receptor . in mice , only cd4 t cells express igd receptors and so cd8 t cells can not participate in the costimulation .
cd4 t cell/ b cell interaction may provide sufficient amplification of rank - l upregulation that the cells triggering osteoclasts are largely restricted to b cells and cd4 t cells .
han et al . have shown that adoptive transfer of antigen - responsive b cells can induce bone loss in rats that are congenitally athymic and therefore t cell deficient .
it is possible that b cells are alone responsible for the effects on bone loss seen in these experiments , but the activation of cd4 t cells over an extended period of time ( 42 days postinfection ; figure 4 ) would argue that t cell activation is playing at least an amplifying role , either through rank - l or through increased secretion of bone resorptive cytokines [ 3 , 20 ] .
genetic deletion of igd was shown here to diminish t and b cell activation and to inhibit alveolar bone loss in mice .
this study suggests a new pathway through which oral infection with p. gingivalis can result in loss of alveolar bone .   in response to oral infection , igd on the surface of b cells
binds to igd receptors on cd4 t cells , resulting in coactivation of both b and t cells of the immune system .
such b and t cell activation can , in turn , activate osteoclasts and trigger bone loss .
chronic periodontitis is a widespread infectious disease ; therefore , elucidation of the mechanisms through which the immune responses to infection trigger damage rather than protection is of clinical importance .
 periodontal disease is one of the most common infectious diseases of humans . 
 immune 
 responses to infection trigger loss of alveolar bone from the jaw and eventual tooth loss . 
 
 we investigated the contribution of b cell igd to alveolar bone loss by comparing the 
 response of b cell normal balb / cj mice and igd deficient balb / c - igh-5/j mice to oral infection with porphyromonas gingivalis , a gram - negative periodontopathic bacterium 
 from humans . 
 p. gingivalis - infected normal mice lost bone . 
 specific antibody to p. 
 gingivalis was lower and oral colonization was higher in igd deficient mice ; yet bone 
 loss was completely absent . 
 infection increased the proportion of cd69 + activated b cells 
 and cd4 + t cells in immune normal mice compared to igd deficient mice . 
 these data 
 suggest that igd is an important mediator of alveolar bone resorption , possibly through 
 antigen - specific coactivation of b cells and cd4 + t cells . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion