Periodontitis and arthritis interaction in mice involves a shared hyper-inflammatory genotype and functional immunological interferences.

Periodontitis (PD) and rheumatoid arthritis (RA) have been found to be clinically associated and to share the chronic nature of the inflammatory reaction associated with bone resorption activity. However, the mechanisms underlying such association are unknown. Therefore, we examined the basis of Actinobacillus actinomycetemcomitans- and Porphyromonas gingivalis-induced PD and pristane-induced arthritis (PIA) interaction in mice. Higher severity PD in the genetically inflammation prone acute inflammatory reactivity maximum (AIRmax) mice strain was associated with higher levels of TNF-alpha, IL-1beta, IL-17, matrix metalloproteinase (MMP)-13, and RANKL, whereas PD/PIA co-induction resulted in even higher levels of IL-1beta, IFN-gamma, IL-17, RANKL, and MMP-13 levels. Conversely, PD/PIA co-induction in AIRmin strain did not alter the course of both pathologies. PIA/PD co-induction resulted in altered expression of T-cell subsets transcription factors expression, with T-bet and RORgamma levels being upregulated, whereas GATA-3 levels were unaltered. Interestingly, PIA induction resulted in alveolar bone loss, such response being highly dependent on the presence of commensal oral bacteria. No differences were found in PIA severity parameters by PD co-induction. Our results show that the interaction between experimental PD and arthritis in mice involves a shared hyper-inflammatory genotype and functional interferences in innate and adaptive immune responses.

A new model of outbred genetically selected mice which present a strong acute inflammatory response in the absence of complement component C5.

OBJECTIVE: Mice selected for a strong (AIRmax) or weak (AIRmin) acute inflammatory response present different susceptibilities to bacterial infections, autoimmune diseases and carcinogenesis. Variations in these phenotypes have been also detected in AIRmax and AIRmin mice rendered homozygous for Slc11a1 resistant (R) and susceptible (S) alleles. Our aim was to investigate if the phenotypic differences observed in these mice was related to the complement system. MATERIAL: AIRmax and AIRmin mice and AIRmax and AIRmin groups homozygous for the resistance (R) or susceptibility (S) alleles of the solute carrier family 11a1 member (Slc11a1) gene, formerly designated Nramp-1. METHODS AND RESULTS: While no difference in complement activity was detected in sera from AIRmax and AIRmin strains, all sera from AIRmax Slc11a1 resistant mice (AIRmax(RR)) presented no complement-dependent hemolytic activity. Furthermore, C5 was not found in their sera by immunodiffusion and, polymerase chain reaction and DNA sequencing of its gene demonstrated that AIRmax(RR) mice are homozygous for the C5 deficient (D) mutation previously described in A/J. Therefore, the C5D allele was fixed in homozygosis in AIRmax(RR) line. CONCLUSIONS: The AIRmax(RR) line is a new experimental mouse model in which a strong inflammatory response can be triggered in vivo in the absence of C5.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis.

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H(III)) or low (L(III)) antibody response and for maximum (AIR(MAX)) or minimum (AIR(MIN)) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR(MIN) mice whereas in the other strains approximately 40% of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

Genetic potential for an acute inflammatory response in IgA glomerulonephritis in mice.

Mice selected on the basis of an acute inflammatory response (AIR) can provide information about the immunopathological mechanisms of glomerulonephritis. We studied the differences between mice selected for a maximal AIR (AIRmax that attract more polymorphonuclear cells to the site of injury) or a minimal AIR (AIRmin that attract more mononuclear cells) in an experimental model of IgA nephropathy in order to investigate the effect of genetic background on glomerular disease progression and the participation of the monocyte chemoattractant protein-1 (MCP-1) chemokine. IgA nephropathy was induced by intraperitoneal ovalbumin injection and bile duct ligation in AIRmax and AIRmin mice. Histological changes, urinary protein/creatinine ratio, serum IgA levels, immunofluorescence for IgA, IgG and complement C3 fraction, immunohistochemistry for macrophages and MCP-1, and MCP-1 levels in macerated kidney were determined. Mesangial IgA deposition was seen only in AIRmin mice, which presented more renal lesions. Increased serum IgA levels (1.5 +/- 0.4 vs 0.3 +/- 0.1 mg/mL, P < 0.001), high glomerular MCP-1 expression and decreased monocyte/macrophage infiltration in the interstitial area (0.3 +/- 0.3 vs 1.1 +/- 0.9 macrophages/field, P < 0.05) were detected in AIRmin mice compared to AIRmax mice. No glomerular monocyte/macrophage infiltration was detected in either strain. In spite of the absence of IgA deposition, AIRmax mice presented discrete or absent mesangial proliferation. The study showed that there are differences between mice selected for AIRmax and AIRmin with respect to serum IgA levels, histological damage and MCP-1 chemokine production after ovalbumin injection in combination with bile duct ligation.

Genetic regulation of the specific and non-specific component of immunity.

Bi-directional selective breeding for antibody (Ab) responsiveness to heterologous erythrocytes (Selection I) produced a high (H) and a low (L) responder line of mice which were also remarkably separated for Ab responses to many unrelated natural antigens (Ags) such as heterologous proteins, viruses, bacteria, parasites and haptens carried by these immunogens. The character "quantitative Ab responsiveness" is controlled by several independently segregating loci (polygenic regulation). The major genetic modification is produced at the level of macrophage activities. The Ag is slowly catabolized and persists for a long time on the macrophage membrane of the H line, whereas it is rapidly destroyed in L line macrophages. The bactericidal and bacteriostatic activity of the macrophage is also strong in the L line and weak in the H line. The opposite genetic regulation of Ab responsiveness and macrophage activity is a fundamental phenomenon for understanding natural and vaccination-induced anti-infectious immunity. The L line is more resistant than the H line against the infections due to intracellular microorganisms (Salmonellae, Yersinia, Mycobacteria, Brucellae, Leishmania) where the macrophage plays the dominant defensive barrier. The H line is more resistant than the L line to the extracellular microorganisms which are efficiently counteracted by a strong antibody response (Pneumococcus, Klebsiella, Plasmodia, Trypanosoma). The intensity of T cell-mediated immunity as measured by delayed type hypersensitivity, which is independent of the genetic regulation of antibody responsiveness, is correlated with the degree of nonspecific inflammation produced at the site of the reaction by the Ag injection in non-sensitized mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Low antibody responsiveness is found to be associated with resistance to chemical skin tumorigenesis in several lines of Biozzi mice.

High and low antibody responder lines of mice from Selections I, III and G were assayed for two-step skin tumorigenesis using a protocol consisting in initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Concordant results were obtained in the three selections: low antibody responder mice were shown to be significantly more resistant to tumor induction than the high responder counterparts. The difference was observed for all parameters: kinetics and percentages of tumor incidence and tumor multiplicity. The three bidirectional selective breeding experiments differed in several respects namely, the origin of the foundation populations, the antigens and immunization protocols used during the selection, as well as the breeding unit environments. Therefore, the consistent results relative to tumorigenesis strongly suggest that some of the alleles relevant to multispecific 'low' antibody production could contribute to the resistance to cutaneous chemical tumorigenesis.

Slc11a1 (formerly NRAMP1) gene modulates both acute inflammatory reactions and pristane-induced arthritis in mice.

Mice selected for the maximum acute inflammatory reaction (AIRmax) are highly susceptible to pristane-induced arthritis (PIA), whereas mice selected for the minimum response (AIRmin) are resistant. These lines show distinct patterns of leukocyte infiltration and R and S allele frequency disequilibrium of the solute carrier family 11a member 1 (Slc11a1) gene. In order to study the interactions of the Slc11a1 R and S alleles with the inflammation modulating Quantitative Trait Loci (QTL) during PIA development, homozygous AIRmax(RR), AIRmax(SS), AIRmin(RR) and AIRmin(SS) lines were produced by genotype-assisted breedings. These mice received two intraperitoneal injections of 0.5 ml pristane at 60-day intervals, and the subsequent development of arthritis was assessed for 210 days. Cytokine-secreting cell profiles were investigated using enzyme-linked immunospot. Arthritis incidence in AIRmax(RR) mice reached 29%, whereas PIA incidence in AIRmax(SS) mice was 70% by day 180. AIRmin(RR) mice were resistant, whereas 13.3% of AIRmin(SS) mice became arthritic. The presence of the defective S allele also increased arthritis severity, although acute inflammation was higher in mice bearing the R allele. A predominant Th0/Th2-type response in Slc11a1(SS) mice was observed. These results indicate that Slc11a1 is a strong candidate for the QTL modulating acute inflammation and for PIA.

Inverse modification of antibody responsiveness to RGG in lines of mice selected for high or low responses to somatic antigen of Salmonella.

High (H/s) and low (L/s) antibody responder lines of mice selected according to their response to the somatic (s) antigen of Salmonella (Selection IV) have unexpected inverse capacity for antibody production to rabbit gamma globulin (RGG): H/s mice are low or even nonresponders to this antigen, whereas L/s mice are high responders. It was shown that the phenotypic variability within each line is due to environmental factors. RGG was a selection antigen in Selection V; the high (H/p) and low (L/p) responder mice are therefore considered as homozygous for the RGG genes. Responsiveness to RGG was investigated in F1 and F2 hybrids obtained by crossing the phenotypically similar RGG responder or nonresponder mice of Selections IV and V. The results support the hypothesis that the same genes control the response to RGG in L/s and H/p lines as well as in H/s and L/p lines. This means that the genes specific for RGG responsiveness were independent from those regulating responses to the s antigen. Unaffected by the selective breeding in Selection IV, they have been fixed by chance in an inverse way in H/s and L/s lines.

Inflammatory cutaneous reaction induced by the lectin of Dioclea grandiflora (Mart.).

The lectin from Dioclea grandiflora (Mart.) that selectively binds glucose and mannose, when subcutaneously injected in mouse induces an inflammatory cutaneous reaction whose histological analysis reveals an hemorrhagic ulceration with exudative reaction accompanied by an influx of polymorphonuclear leukocytes and giant cells. The presence of lymphocytes and plasma cells in the lesion was insignificant. In order to characterize the in vivo action of inflammatory factors generated by this lesion, distinct lines of mice were used: high and low antibody responder mice; the genetically selected mice to the acute phase of inflammatory reaction; lines of mice deficient in C5, a protein of the complement system. It is shown that the lectin of D. grandiflora acts as an inflammatory agent probably promoting exocytosis and release of mediators.

Non-specific regulation of immune responsiveness to a thymus-independent antigen, TNP-LPS, in high and low responder lines of mice.

Responsiveness to a thymus-independent (TI-1) antigen, TNP-LPS, was investigated in the high and low responder lines of mice resulting from four independent selective breedings carried out for antibody production to various complex natural immunogens (selections I, III, IV and V). The superiority of the high responder vs. the low responder line was generally observed, confirming that the genes accumulated through selective breeding can modify the responsiveness to unrelated antigens including TI antigens. Two special features were observed in selection III: (1) A secondary response to TNP-LPS: higher peak values and IgG isotype antibody production were obtained in the H line. (2) Pretreatment with LPS modified the responses to TNP in the L line only.

Independent polygenic regulation of quantitative antibody responsiveness and expression of delayed-type hypersensitivity (DTH).

The intensity of delayed-type hypersensitivity (DTH) expression measured by footpad swelling was established in high and low antibody responder lines of mice produced by bidirectional selective breeding for antibody responsiveness to different antigens. These lines of mice presented a very large difference in antibody response to the antigens used in each selective breeding (selection Ags) and to several other unrelated Ags (nonspecific effect). The intensity of DTH reactivity to selection Ags and to unrelated Ags differed in the various lines investigated, but the intensity of DTH reactions was not correlated with antibody responsiveness. The results of the present article demonstrated that the expression of DTH reactivity and antibody responsiveness to the same antigens are polygenic characters subject to independent quantitative regulation.

T-helper functions in lines of mice selected for high or low antibody production (Selection III): modulation by anti-CD4+ monoclonal antibody.

T-helper function was evaluated in mice genetically selected for high (H) or low (L) antibody (Ab) responsiveness to Salmonella flagellar antigen (Ag) (Selection III). In this Selection as opposed to what was demonstrated in Selections I, II and IVA, the interline difference was not proven to be based upon the modification of Ag processing and presentation at macrophage level. CD4+/CD8+ lymph node ratio is similar in HIII and LIII mice, both lines being equally susceptible to in vivo depletion of CD4+ T cells by GK 1-5 monoclonal antibody (mAb) treatment. Nevertheless, the Ab responsiveness of the two lines was differently modulated by GK 1-5 mAb: the inhibition of Ab responses to various Ag required lower mAb doses and was long lasting in LIII as compared to the transient effect of higher mAb doses observed in HIII. LIII mice were also refractory to Salmonella-induced reversion of GK 1-5 mAb inhibition. Moreover, in vitro specific I proliferation was constantly lower in LIII, though its IL-2 production was unexpectedly similar to that of HIII T cells. Results of in vivo and in vitro experiments are thus consistent with a defective response of T-helper cells to immunogenic challenge in LIII mice.

Genetics of acute inflammation: inflammatory reactions in inbred lines of mice and in their interline crosses.

Acute inflammation is induced by the subcutaneous injection of swollen polyacrylamide microbeads, its intensity measured by the cell and protein concentration of the local exudates. A large and continuous range of responses is obtained in different inbred strains of mice, which suggests a polygenic control of the inflammatory response. The variable levels of the global dominance observed in F1 hybrids issued from several parental combinations indicated that the pattern of alleles controlling high or low response was different in each parental strain. Balanced intercrossing of the 8 inbred strains studied has provided a genetically heterogeneous F3 population, presenting a high variability of responses. The value of the genetic part of F3 phenotypic variance, the spread of the interstrain differences, as well as the polygenic nature of the regulation of inflammatory responses pointed out the possibility to perform a bidirectional genetic selection by using the F3 mice as the foundation population, and response to microbeads as the selective phenotypic character.

Polygenic control of quantitative antibody responsiveness: restrictions of the multispecific effect related to the selection antigen.

Among the differences observed between the various high (H) and low (L) antibody responder lines of mice resulting from distinct bidirectional selective breedings, one of the most puzzling is the variation in the "multispecific effect," i.e., in the modification of antibody responses to antigens unrelated to those used during the selection. The best examples are the H and L lines of selection IV, selected on the basis of responses to somatic antigen of Salmonella which do not differ in their antibody responses to sheep erythrocytes (SE). However, a wide range of variability is observed in the responses of (HIV X LIV)F2 hybrids to this antigen, and it was therefore hypothesized that distinct groups of genes might regulate antibody responses to SE and the somatic antigen. Indeed, a new selection (IV-A) for anti-SE responsiveness started from these (HIV X LIV)F2 successfully produced a high and a low anti-SE responder line. The results of selection IV-A and the variance analysis of (HIV-A X LIV-A)F2 hybrids are reported. They are roughly similar to those in selection I, also carried out for anti-SE responsiveness. In vivo attempts to identify the major regulatory mechanism which contributes to the interline difference indicate that the efficiency of macrophage accessory function has been modified in selection IV-A, as was observed in selection I, whereas this function did not differ in HIV and LIV lines. Probably in relation to the involvement of macrophage function there is a notable increase of the multispecific effect in selection IV-A when compared with selection IV. The results of selection IV-A demonstrate that responsiveness to heterologous erythrocytes and to somatic antigen of Salmonella are under separate polygenic control operating through distinct regulatory mechanisms. The choice of the selection antigen and immunization procedure is of major importance for defining the gene interaction operating in each selective breeding experiment and the extent of its multispecific effect.

Basal immunoglobulin serum concentration and isotype distribution in relation to the polygenic control of antibody responsiveness in mice.

Serum Ig concentration and isotype distribution were determined in the high (H) and low (L) responder lines selected for antibody response to complex immunogens. Data were recorded in normal and postimmunization sera from the H and L lines produced by five independent selective breedings (selections I, II, III, IV, and V). Ig levels were much higher in H than in L mice of all the selections. In four selections this interline difference increased further after immunization with the selection antigens. This is in agreement with the general effect of the polygenic control of antibody responses operating in H and L lines. The Ig isotype profiles of normal sera were different in each line; however, similitudes were noticed between H and L lines in selections I and II. In contrast, in selections III, IV, and V a similar interline difference was observed: the lack of IgG2a isotype in L lines only. After immunization there were minor alterations of the isotype profiles except in the H lines of selections III and IV, in which a clear inverse modification of IgG1 and IgG2a proportions occurred. The characteristic pattern of each selection may be partially dependent on isotype-restricted regulatory effects in relation to the immunization procedure used for selective breeding.

Effect of silica on the genetic regulation of antibody responsiveness.

The high (H) and low (L) antibody responder lines of mice produced by selective breeding are characterized by different modifications in immunocompetent cell potentialities, according to the immunization procedure used for the selection process. In selections I and II, the difference in antibody responsiveness between H and L lines was clearly shown to depend mainly on macrophage function: the more rapid catabolism of antigens in L mice was the main cause of the low antibody production. In contrast, up to now, no difference has been observed between H and L mice of selections III and IV in terms of the macrophage accessory role. The administration of silica particles has a well known impairment effect on macrophage activity. Therefore, the effect of silica injection on the kinetics of antibody responses to selection antigens was compared in H and L mice of the four selections. Silica was given either intravenously or locally in one hind footpad 6 or 24 h before immunization by the same route. Silica treatment consistently improved antibody responsiveness in the L mice of selections I and II, but had no effect in the L mice of selections III and IV. The antibody responses of the H lines of the four selections were not substantially modified by silica injections. Therefore, the silica treatment reduced the interline difference in antibody responses in selections I and II only, by interfering with the expression of the genetic modification of macrophage activity. However, a similar effect was not obtained with other substances known to affect macrophages, including dextran sulphate or carrageenan. The results reported here are in agreement with the above-mentioned statement that the genetic modification of macrophage function plays a major role in the interline difference in selections I and II and is not involved in selections III and IV.

Nonspecific genetic regulation of antibody responsiveness in the mouse.

Four lines of mice were produced by selective breeding for quantitative agglutinin responsiveness to flagellar (f) or somatic (s) antigens (Ags) of Salmonellae: high (H) or low (L) responder lines to fAg and H and L responder lines to sAg. The Salmonellae contained both f and sAgs, the Ag used to perform the selection was the Selection Ag and the other was the Associated Ag. The selective breeding produced a progressive interline separation with an equivalent effect for both Ags. After 15 generations (F15) the level of agglutinin response was about 60 times higher in H than in L responders. About 50% of the phenotypic variation of the character investigated is determined by a group of immune response genes, the rest is due to environmental factors. The nonspecific effect of this group of immune response genes was investigated by measuring the responses to three independent antigens: Sheep erythrocytes (SE), dinitrophenyl-conjugated human IgG (DNP-HGG) and bovine IgG (BGG). The selection for fAg response produced an equivalent modification in the respnsiveness to the Associated Ag (97%) and to BGG (130%). This nonspecific effect was smaller for responsiveness to SE and DNP-HGG, 58% and 41% of the Selection Ag response, respectively. The selection for sAg response produced a nonspecific modification of responsiveness of 94% for the Associated Ag of 74% for BGG and 63% for DNP-HGG. An important exception concerned SE to which an equal antibody response is produced in high and low lines of sAg selection.

Salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection.

Susceptibility to Salmonella typhimurium infection was compared in H (high Ab responder) and L (low Ab responder) mice obtained by several selective breeding experiments (Selections I, II, III, IV and IV A). H mice were always much more susceptible to infection than their L mice counterparts within a continuous LD 50 variation range. In three of the selections (I, II and IV A) the low responsiveness character is known to result mainly from rapid Ag degradation in L mice macrophages. It was hypothesized that resistance to multiplication of intracellular pathogens could be related to an increased catabolic activity towards Ag. This was actually demonstrated, in F2 segregant hybrids of selection IV A, by the significant inverse correlation between capacity for Ab production and resistance to infection.

Innate resistance to infection by intracellular bacterial pathogens differs in mice selected for maximal or minimal acute inflammatory response.

The intensity of nonspecific immune reaction and the host resistance to facultative intracellular pathogens are found to be associated in lines of mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactivity. AIRmax are more resistant than AIRmin mice to Salmonella typhimurium and Listeria monocytogenes infection, the differences between lines in LD50 being > 1000 and 100 times, respectively. This difference was shown to be related to the initial bacterial containment at the infectious focus, and to the control of bacterial multiplication in the spleen during the 1st week after s. c. inoculation of the bacteria. Specific immune responses were not deeply affected by the selective process: antibody production and delayed-type hypersensitivity were both of similar intensity in AIRmax and AIRmin mice. The differential susceptibility to infection seems independent of the Nramp-1 locus polymorphism; therefore, these two lines represent a powerful model for investigating the role of other genetic loci regulating the nonspecific immunity effectors in the course of infectious diseases.