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Keywords:

  • Autoantibodies;
  • Autoimmunity;
  • Chemokines

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Development of autoimmunity is a multi-factorial process involving genetic predisposition as well as environmental and stochastic factors. Although the mechanisms responsible for the initiation of autoimmunity remain only partially understood, several studies have demonstrated that genetic predisposition plays a major role in this process. In the present study, we analyzed the influence of CCR7 signaling in the development of autoimmunity, because this chemokine receptor is essentially involved in the functional organization of thymus architecture. We demonstrate that CCR7-deficient mice are prone to develop generalized multi-organ autoimmunity. The autoimmune phenotype of CCR7–/– mice encompasses the presence of lymphocyte infiltrates in several peripheral organs, circulating autoantibodies against a multitude of tissue-specific antigens and IgG deposition on renal glomeruli. Additionally, CCR7-deficient mice show increased susceptibility to streptozotocin-induced diabetes and spontaneously display signs of chronic autoimmune renal disease. Thus, this study identifies CCR7 as a genetic factor involved in the regulation of autoimmunity.

Abbreviations:
ANA:

anti-nuclear antibodies

SPF:

specific pathogen-free

STZ:

streptozotocin

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

A major challenge of the immune system is to efficiently combat infectious pathogens while maintaining self-tolerance and avoiding immune-mediated pathologies. In order to meet this complex challenge, structural compartmentalization of the immune system has evolved during evolution in higher vertebrates. Several studies have underlined the importance of the chemokine system as a major organizer of the immune system coordinating proper development, activation and function of distinct leukocyte populations 13. In particular, the chemokine receptor CCR7 and its ligands, CCL19 and CCL21, have been identified to essentially participate in various processes required for the maintenance of a functionally intact immune system. These processes include development and organization of secondary lymphoid organs, initiation of immune responses and development of oral tolerance 47. An important role for CCR7 in thymus compartmentalization and orchestration of migratory events during thymopoiesis has also been recently demonstrated 8, 9.

The essential function of CCR7 in the organization and maintenance of immune system integrity is well illustrated in mice deficient in CCR7–/– or for its ligands CCL19 and CCL21-ser (plt/plt mice) 10, 11. In these animals, several immunological disorders have been reported, including altered thymus architecture 8, 9. Interestingly, thymus architecture is frequently affected in mice suffering from autoimmunity such as mice deficient in the transcriptional regulator autoimmune regulator (Aire) and non-obese diabetic (NOD) mice 12, 13. Therefore, the relevance of CCR7 signaling in the maintenance of thymus function raised the question whether CCR7-deficient animals are prone to autoimmunity. It has been noticed by others that CCR7–/– and plt/plt mice manifest mild autoimmunity to the lachrymal and salivary glands 14. However, it remains unclear whether lack of CCR7 signaling leads to a general propensity for the development of autoimmunity and more importantly for the onset of autoimmune diseases.

In the present study, we demonstrate that CCR7-deficient mice in fact display not a localized but a generalized autoimmune phenotype that is not restricted to the salivary and lachrymal glands but affects several organs, including pancreas and stomach. In line with this observation, CCR7–/– mice harbor increased titers of circulating autoantibodies to nuclear and non-nuclear tissue-antigens and display IgG deposits on renal glomeruli. Despite the presence of autoimmunity to the salivary glands and pancreas CCR7-deficient mice developed neither Sjögren's syndrome-like disease nor diabetes spontaneously. However, they show increased susceptibility to streptozotocin (STZ)-induced diabetes as well as manifestations of ongoing spontaneous autoimmune renal disease. Therefore, data presented here demonstrate that CCR7-deficient mice are prone to the development of autoimmunity, although autoimmunity not always results in the onset of disease.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Lymphocytic infiltration in peripheral organs of CCR7–/– mice

Similar to mice suffering from spontaneous autoimmune disorders, CCR7–/– mice display an altered thymic architecture 8, 9. Therefore, it seemed possible that lack of CCR7 signaling might result in development of autoimmunity. A hallmark of mice prone to autoimmunity is the presence of lymphocytic infiltration in peripheral organs. Indeed, a histological analysis of several organs of 5- to 7-week-old specific pathogen-free (SPF) CCR7-deficient animals revealed lymphocyte infiltrates in the lachrymal gland, stomach, lung, submandibular gland, pancreatic islets and liver (Fig. 1A and data not shown). Infiltrates were observed in the organs of all mice analyzed independently of their gender (Table 1). Additionally, the histological analysis of germ-free CCR7–/– mice demonstrated that the presence of infiltration in peripheral tissues is not due to exacerbated T cell responses to microbial antigens (Table 1). Interestingly, the infiltrates observed in the stomach and lachrymal glands of germ-free mice were often more pronounced than those found in SPF mice. Some of the germ-free analyzed animals displayed massive infiltration of the lachrymal gland with complete destruction of the glandular tissue (Fig. 1B). Such a complete destruction of the lachrymal gland was never observed in the SPF analyzed animals. Additionally, in contrast to SPF animals, approximately 40% of the analyzed germfree mice showed infiltrations in the parotid gland (Table 1), indicating that organs of CCR7-deficient mice are even more susceptible to the presence of infiltrates in the absence of microbial antigens. Together, the results suggest that the presence of infiltration in peripheral organs of CCR7-deficient animals is due to reactivity to self-antigens.

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Figure 1. Lymphocytic infiltration in peripheral organs of CCR7–/– mice. (A) Prominent lymphocytic infiltrations are present in lachrymal and submandibular glands, stomach and pancreatic islets of CCR7–/– mice. Hematoxylin and eosin staining of representative sections from peripheral organs of 5- to 7-week-old SPF wild-type (+/+) and CCR7-deficient (-/-) mice are shown, arrows indicate infiltration foci. Note the presence of lymphoid aggregates in the gastric mucosa. (B) Presence of exacerbated lymphocyte infiltrates in the lachrymal gland and stomach of germ-free CCR7-deficient mice.

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Table 1. Lymphocyte infiltrates in SPF and germ-free CCR7-deficient animals
HousingconditionsGenderAge(weeks)Organ
SubmandibularParotidLachrymalPancreasStomach
SPFMale5–79/90/99/99/99/9
Female75/50/55/55/55/5
Germ-freeMale9–104/42/44/44/44/4
Female6–104/41/44/43/34/4

Presence of organized tertiary lymphoid structures in the stomach of CCR7–/– mice

Tertiary lymphoid structures have been observed in affected organs of patients suffering from autoimmune diseases such as Sjögren's syndrome, multiple sclerosis, rheumatoid arthritis and autoimmune thyroiditis. During the histological analysis of peripheral organs of CCR7–/– animals we observed the presence of lymphoid aggregates in the gastric mucosa and submucosa (Fig. 1A, arrows). Immunofluorescence staining on stomach sections of CCR7-deficient mice revealed these structures to be highly organized, displaying discrete T and B cell areas, and containing CD11c+ DC (Fig. 2A). In addition, we also observed high endothelial venules (HEV) expressing the peripheral node addressin (PNAd) within these lymphoid aggregates (Fig. 2B, arrows). Therefore, the lymphoid aggregates observed in the stomach of CCR7–/– mice resemble tertiary lymphoid organs found in affected organs in autoimmune diseases. To prove whether presence of tertiary lymphoid organs in the gastric mucosa and submucosa of CCR7-deficient mice correlates with an ongoing autoimmune response, we analyzed the presence of circulating antibodies to gastric-derived antigens in the serum of these animals. As shown in Fig. 2C and D serum from individual CCR7–/– mice (five of seven analyzed animals) but not from wild-type controls reacts with the cytoplasm of parietal cells in the gastric mucosa. In addition, serum of some CCR7-deficient mice but of none of the control mice reacts with the nuclei of the cells resulting in a complete overlay with the 4′6′-diamino-2-phenylindole (DAPI) nuclear stain (Fig. 2E and F). No gender-related differences have been observed in this experiment. Enzyme-linked immunosorbent assay (ELISA) confirmed that CCR7–/– mice have substantially increased titers of IgG to gastric-derived antigens compared to age-matched controls (Fig. 2G). Importantly, no significant differences regarding the titers of these antibodies were observed between animals kept under specific pathogen-free (SPF) conditions and germ-free mice (Fig. 2H). This result indicates that these antibodies are not direct to microbial antigens but to self-antigens. Furthermore, the nuclear stain obtained with the sera of CCR7-deficient mice indicates the presence of circulating anti-nuclear antibodies (ANA) in these animals. Antibodies specific for DNA are a major component of ANA and anti-DNA antibodies are often used as a diagnostic marker for systemic autoimmunity 15. Therefore, we further analyzed the sera of mutant and control mice for the presence of antibodies directed to dsDNA by ELISA. As shown in Fig. 2I, both SPF and germ-free CCR7-deficient mice display significantly increased titers of anti-dsDNA antibodies compared to SPF wild-type mice, even though the antibody titers were higher in SPF than in the germ-free mice. Together, the results demonstrate that an autoimmune response occurs in CCR7-deficient mice.

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Figure 2. Ongoing immune response to gastric antigens in CCR7-deficient mice. (A) Lymphoid aggregates in the gastric mucosa of CCR7–/– mice were analyzed by immunofluorescence staining with antibodies to CD11/c (green), B220 (blue) and CD4 (red). Note that the aggregates are organized in discrete B cell (blue) and T cell (red) areas. (B) Presence of HEV (high endothelial venules) in lymphoid aggregates of the gastric mucosa. Endothelial vessels were stained with antibodies to PECAM-1 (green) and PNAd (red) within the lymphoid aggregates (blue). (C, D) Serum of CCR7-deficient but not of wild-type mice reacts with the cytoplasm of parietal cells (green), indicating auto-reactivity to gastric antigens. Nuclei are stained with DAPI (blue). (E, F) Serum of CCR7–/– mice also reacted with the nuclei of cells in the gastric mucosa, indicating the presence of circulating anti-nuclear-antibodies in these mice. For all stainings, cryosections of the stomach of wild-type mice were incubated with mouse serum diluted 1:100. Binding of circulating IgG to the tissue was revealed with a rat anti-mouse-FITC antibody. (G) Serial dilution of serum from wild-type (+/+) and CCR7–/– (-/-) mice (n = 8) revealed increased titers of circulating IgG to gastric antigens in the mutant mice. (H) Comparable titers of antibodies to gastric antigens were found in SPF and germ-free mutant mice (n = 10). For this assay, serum at a dilution of 1:270 was used. ELISA plates in (G) and (H) were coated with stomach tissue lysate from wild-type mice. (I) Increased titers of circulating IgG to dsDNA in SPF (n = 22) and germ-free (n = 14) CCR7–/– mice in comparison to SPF (n = 22) wild-type mice. ELISA plates were coated with salmon sperm DNA. For this assay, mouse sera were diluted 1:150.

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Autoimmunity to salivary and lachrymal glands in CCR7–/– mice

As shown in Fig. 1, CCR7-deficient mice exhibit lymphocytic infiltration in the submandibular and lachrymal glands. Immunofluorescence revealed the presence of diffuse infiltrates as well as lymphoid aggregates containing discrete T and B cell areas in the glands of 5–7-week-old CCR7–/– mice (Fig. 3A). T and B cell infiltrations in exocrine glands are characteristic of Sjögren's syndrome, a human autoimmune disease that frequently leads to the destruction of salivary and lachrymal glands. Because CCR7-deficient mice exhibit histological features characteristic for Sjögren's syndrome, we further analyzed these animals for the presence of serum antibodies to glandular antigens. As shown in Fig. 3B, CCR7–/– mice possess increased titers of antibodies to antigens derived from the submandibular gland in comparison to wild-type mice. More importantly, compared to age- and sex-matched controls, young CCR7-deficient mice showed significantly elevated titers of antibodies to alpha-fodrin (Fig. 3C). Furthermore, anti-alpha fodrin antibody titers exceeding the mean + 3 SD of the titers found in the wild-type group (OD values above 0.187) were detected in 60% of the analyzed CCR7-deficient mice (Fig. 3C). Absorbance values greater than the mean + 3 SD of the control group has been considered as seropositive for alpha-fodrin in an earlier study 16. Alpha-fodrin has been recently identified as an antigen associated with Sjögren's syndrome and the presence of elevated titers of circulating antibodies to this protein correlates in humans with onset of disease 17. It is of note that the titers of anti-alpha-fodrin antibodies increased with age in both mutants and wild-type mice, with the titers being permanently higher in mutants. Consequently, 94% of the old mutants and 69% of the age-matched wild-types analyzed were seropositive for alpha-fodrin (Fig. 3C). The increased titers of anti-alpha-fodrin antibodies observed in old wild-type mice is most probably caused by factors associated with immune senescence such as decreased capacity for tolerance-induction 18. Gender specific differences have not been observed with respect to anti-alpha-fodrin antibody titers, wherefore values for males and females are not displayed separately in Fig. 3C. Despite the histological and serological signs of Sjögren's syndrome, clinical manifestation of the disease was not observed in young CCR7-deficient mice as assessed by measurement of their saliva secretory function after pilocarpine hydrochlorid injection, a drug used to stimulate saliva production (Fig. 3D). In aged CCR7–/– mice a slight reduction in the production of saliva could be observed in comparison to age-matched wild-type animals. These results clearly demonstrate the presence of self-reactivity to glandular tissue antigens in CCR7-deficient mice yet without showing overt signs of severe disease.

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Figure 3. Autoimmunity to salivary and lachrymal glands in CCR7–/– mice. (A) Cryosections of the salivary and lachrymal glands of CCR7-deficient mice were stained with antibodies to B220 (red) to identify B cells and CD3 (blue) to identify T cells. Note the presence of diffuse infiltrates as well as organized lymphoid aggregates. (B) Increased titers of circulating IgG to salivary gland-derived antigens in the serum of CCR7-deficient (-/-) in comparison to wild-type (+/+) animals (n = 7). ELISA plates were coated with a lysate of salivary glands from wild-type mice. (C) Presence of increased titers of anti-alpha-fodrin IgG in the serum of young (6- to- 8 weeks; n = 18; p <0.0001) and aged (>26 weeks; n = 16; p = 0.0235) CCR7–/– mice. For this assay, mice sera were diluted 1:150. The dashed line indicates the OD value 0.187 that corresponds to the mean + 3 SD of the control group. (D) Saliva secretion assay of young and old mice. The volume of saliva was determined after pilocarpine stimulation. Values were normalized to body weight. No significant differences were found between CCR7-deficient and wild-type mice in both groups, although in aged mutant mice the saliva secretion was slightly decreased.

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Increased susceptibility to the induction of autoimmune diabetes in CCR7–/– mice

CCR7-deficient mice show mild to moderate lymphocytic infiltration in the periphery of pancreatic islets (Fig. 1A). This type of infiltration is commonly associated with ongoing inflammatory autoimmune responses in the pancreatic islets known as insulitis, which culminates in the destruction of β-cells prior to the onset of diabetes. This sub-clinical phase of the disease is accompanied by presence of serum antibodies to islet antigens 19. To test whether presence of peri-islet infiltrates in CCR7-deficient mice correlates with an ongoing immune response, we tested the presence of circulating antibodies to islet antigens by indirect immunofluorescence on pancreas sections of Rag1–/– mice. Serum of CCR7–/– mice stained pancreatic islets, indicating the presence of circulating antibodies to islet antigens (Fig. 4A, arrows). Again, the prominent staining of cell nuclei demonstrates the presence of ANA in the serum of these animals (Fig. 4A).

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Figure 4. Increased susceptibility for the development of diabetes in CCR7-deficient mice. (A) Presence of antibodies to pancreatic islet antigens in the serum of CCR7-deficient animals. Cryosections of the pancreas from Rag1–/– mice were stained with serum of wild-type (+/+) and mutant (-/-) mice as described in Fig. 2. Serum stain is shown in green and nuclear DAPI stain in blue. Serum of CCR7-deficient mice also stained the nucleus of the cells confirming the presence of circulating anti-nuclear antibodies in the serum of these mice. (B) Increased titers of anti-insulin IgG were observed in the serum of young (6 to 8 weeks) CCR7–/– animals (-/-). Note that increased titers were more prominent in females. (C) Despite increased titers of antibodies to pancreatic islet antigens normal blood glucose levels were found in young CCR7-deficent males and females. (D) Increased susceptibility to STZ-induced diabetes in CCR7–/– mice. Wild-type (+/+) and mutant (-/-) males (n = 10 for each group) were injected with multiple low doses of STZ and killed directly after detection of disease.

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Insulin is a primary auto-antigen causing diabetes in NOD mice 20. As revealed by ELISA, 6 to 10 week old CCR7–/– mice have increased titers of circulating anti-insulin antibodies compared to age matched controls (Fig. 4B), with the difference between mutant and wild-type animals being more pronounced in females. Presence of insulitis and elevated titers of circulating antibodies to insulin indicate that CCR7–/– mice might carry an increased predisposition for the development of diabetes. However, as only 30 to 40% of the pancreatic islets from 5–8-week-old CCR7–/– mice show peri-islet infiltration and due to the fact that onset of diabetes is not observed unless approximately 90% of the β-cells have been destroyed 19, we did not expect symptoms of diabetes in CCR7-deficient mice. Indeed, we did not observe any differences regarding blood glucose levels between CCR7–/– and wild-type animals (Fig. 4C). Therefore, we treated wild-type (Charles River) and mutant animals with multiple low doses of streptozotocin (STZ) to determine whether CCR7-deficient mice show increased susceptibility to disease. Induction of diabetes by low-dose application of STZ is a frequently used murine model for type-1-diabetes in which responses to self-antigens are crucial for the pathogenesis of the disease 21, 22. As expected, STZ treatment induced diabetes in both, wild-type and CCR7-deficient mice. However, the onset of diabetes was accelerated and the incidence of disease was increased among CCR7-deficient animals (Fig. 4D). Therefore, the data indicate that CCR7-deficient mice have an increased propensity for the development of autoimmune diabetes.

Spontaneous systemic autoimmune disease in CCR7-deficient animals

The presence of ANA is a hallmark of systemic autoimmune diseases such as systemic lupus erythematosus (SLE) in humans and mice. SLE can affect multiple organs such as skin, lung, brain, joints, heart and kidney. Renal dysfunction is thought to be initiated by the accumulation of immunoglobulin complexes in the glomeruli 23. Since ANA were detected in 85% of all analyzed CCR7-deficient mice older than 16 weeks we applied immunofluorescence to test for the presence of glomerular IgG deposits. In fact, pronounced IgG deposits were present in the majority of the glomeruli of all analyzed CCR7-deficient males (Fig. 5A and B). In sharp contrast, only one out of eight CCR7-deficient females analyzed carried glomerular IgG to an extent comparable to that observed in males (Fig. 5B). IgG deposits were never detected in wild-type males whereas similar to the data obtained for mutant females one out of eight analyzed wild-type females scored positive for them. Notably, only the minority of the glomeruli was affected in the later female (Fig. 5B). To investigate possible functional consequences of glomerular IgG deposits, we determined protein concentrations in the urine and checked for creatinine clearance in CCR7–/– and control animals. As depicted in Fig. 5C, CCR7–/– mice showed significantly increased protein concentrations in urine. In addition, creatinine clearance was significantly reduced in these animals (Fig. 5D). Taken together the data indicate that CCR7–/– animals, in particular young males, spontaneously develop chronic renal autoimmune disorder which shares similarities with SLE-associated glomerulonephritis.

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Figure 5. Spontaneous chronic renal autoimmune disease in CCR7-deficient mice. (A) Deposition of IgG complexes on the glomeruli of CCR7–/– mice. Cryosections of the kidney of wild-type (+/+) and mutant (-/-) mice were stained for IgG deposits with an anti-mouse IgG-FITC antibody (green). Glomeruli were stained with Alexa Fluor®555 Phalloidin (red). (B) IgG deposits were found in all CCR7–/– but in none of the wild-type males analyzed. Although no differences in the number of females showing glomerular IgG deposits have been observed between both genotypes (one each), a much larger proportion of glomeruli were affected in the CCR7-deficient female. (C) Increased protein concentrations in the urine of young CCR7–/– mice in comparison to age-matched wild-type mice (for males n = 24, p <0.0001; for females n = 10, p = 0.049). (D) Creatinine clearance in urine of the same animals. Creatinine clearance was estimated by calculating the ratio of protein to creatinine in the same urine sample. The increased protein/creatinine ratio found in CCR7-deficient animals indicates decreased creatinine clearance (for males p <0.0001; for females p = 0.0012). Data presented here are derived from two independent experiments. For one experiment control males (n = 12) were obtained from Charles River.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

CCR7 is a major regulator of the immune system orchestrating a broad spectrum of fundamental processes ranging from lymphoid organ development to induction of defensive immune responses as well as oral tolerance 35, 7. In the present study, we demonstrate that CCR7-deficient mice are marked by lymphocyte infiltrates in peripheral organs. These infiltrates were also found in germ-free CCR7–/– mice, indicating that they were provoked by endogenous factors such as responses to self-antigens rather than by reactions to external immunological insults. The presence of infiltrates accompanied by tissue damage has been previously observed in the salivary and lachrymal glands of CCR7–/– and plt/plt mice 14. This phenotype has been clearly associated with autoimmunity, as adoptive transfer of either thymocytes or splenocytes of plt/plt animals into non-irradiated RAG2-deficient mice results in the accumulation of lymphocytes in these glands. Since the authors did not find infiltrates in the other analyzed organs (trachea, thyroid, liver, spleen, kidney, intestine, adrenal gland and ovary), they concluded that the autoimmune phenotype of CCR7-deficient mice is restricted to the salivary and lachrymal glands. However, Kurobe and colleagues seemingly did not investigate the presence of lymphocyte infiltrates in the lung, stomach and pancreas of mice deficient in CCR7. In accord to the previously published data, we also failed to observe obvious infiltrations in the trachea, thyroid, kidney and adrenal glands of CCR7-deficient animals (data not shown). In contrast, we noticed mild perivascular infiltrates in the liver of the mutant animals analyzed. Therefore, the histological analysis performed in this study demonstrates that the spectrum of affected organs is broader than originally supposed.

During the submission process of this manuscript, Hoepken and coworkers 24 also reported the presence of lymphoid aggregates in the lung, stomach and colon of CCR7-deficient animals. The lymphoid aggregates observed in this study are morphologically very similar to the tertiary lymphoid structures described by us. Interestingly, the gastric lymphoid aggregates analyzed by Hoepken and colleagues contained high numbers of effector memory T cells expressing high levels of CD44 and low levels of CD62L. Based on this observation, the authors suggested that CCR7 is required for the egress of effector memory T cells from the mucosal tissues into the circulation. Nevertheless, they did not exclude the possibility that CCR7 deficiency might lead to breakdown of central tolerance and consequently to the development of autoimmunity as the analyzed mice suffered from hypertrophic gastropathy 24. In fact, it has been recently demonstrated that CCR7 signaling guides lymphocyte exit from peripheral tissues 25, 26. Therefore, it would be possible that impaired egress of lymphocytes from the peripheral tissues contributes to the phenotype observed in CCR7-deficient animals. However, the lymphocyte infiltrates observed by us were confined to particular structures rather than being distributed indiscriminately throughout the affected organs. For example, lymphocyte accumulation was restricted to the perivascular regions of the submandibular glands and to the β-cell islets in the pancreas, indicating that the infiltrates are not merely caused by the accumulation of cells in these organs, but result from the reactivity of infiltrating cells to specific substructures. More importantly, we noticed a significant increase in serum levels of autoantibodies in CCR7-deficient animals. As demonstrated by immunostaining, the antigenic repertoire recognized by these antibodies ranged from nuclear constituents to unknown, yet specifically expressed, components of the gastric mucosa and pancreatic islets. Immunostains were not simply elicited by unspecific pan-reactivity as evidenced by the presence of IgG antibodies specifically recognizing alpha-fodrin, insulin and dsDNA. Additionally, there is a good correlation between the presence of infiltrates and elevated titers of antibodies reacting to components of the affected organs. In some experiments, we discriminated between male and female animals because it is well recognized that autoimmune diseases may show a sex-specific preponderance. Whereas lymphocyte infiltrates were found to occur in a gender-independent manner, the titers of autoantibodies differed between males and females depending on the antigen analyzed, as observed in the case of antibodies specific for insulin. The significance of this finding remains unexplained so far but illustrates the multi-facetted as well as multi-factorial setting in the establishment of autoimmunity. This particular scenario renders a clear-cut genotype to phenotype relationship difficult to uncover. Indeed, there is no apparent link to CCR7 deficiency, direct or indirect in nature, explaining why insulin-specific autoantibodies should be preferentially produced by females, whereas males are primarily affected by the deposition of immune complexes in the kidney. Although genetic predisposition is the predominant factor determining the evolution of autoimmunity, environmental and stochastic factors can strongly influence this process 27. Therefore, it seems possible that environmental factors are contributing to the differences observed between CCR7-deficient animals kept under SPF or germ-free conditions. In accordance with this hypothesis, it has been reported by others that NOD mice housed under SPF conditions develop diabetes more frequently than mice housed in conventional caging, indicating an increased risk for disease development when microbial infections are limited 28. Therefore, it seems reasonable that presence or absence of microbial antigens as well as stress generated by the maintenance of males in a shared cage influence the manifestation of autoimmunity in CCR7-deficient mice.

Although the presence of infiltrates in peripheral organs and increased levels of circulating antibodies to self-antigens are hallmarks of autoimmunity, they do not necessarily correlate with overt autoimmune disease. CCR7–/– mice show some signs of ongoing autoimmune disease, such as the presence of ectopic lymphoid structures in the gastric mucosa and salivary glands as well as disturbed renal functions. However, despite an existing auto-reactivity to salivary gland and pancreatic islet antigens, CCR7-deficient mice spontaneously developed neither a severe Sjögren's syndrome-like disease nor diabetes even at an advanced age.

Sjögren's syndrome has been extensively studied in NOD and MRL/lpr mice, although NOD mice are primarily used as a model for autoimmune diabetes and MRL/lpr mice in models for SLE. More recently, mice deficient in the transcription factor Id3 have been described as a suitable model for Sjögren's syndrome 29. Similar to CCR7-deficient mice, all these strains show histological and serological manifestations resembling the human disease. However, only NOD and Id3-deficent mice and not MRL/lpr mice exhibit a decrease in tear and saliva secretion. At least in NOD and MRL/lpr mice, multiple genetic loci contribute to the susceptibility to develop symptoms of disease and the genetic background plays an important role in disease progression 30. Therefore, autoimmunity to the lachrymal and salivary glands does not necessarily correlate with the onset of Sjögren's syndrome. Additionally, severe infiltrates were observed neither in the submandibular gland nor in the parotid gland of young CCR7-deficient SPF mice, indicating that the salivary glands of these animals are still able to produce normal amounts of saliva. Although saliva production was strongly reduced in some aged CCR7-deficient animals no clear correlation to salivary gland infiltrates could be observed since age-matched controls also show reduced saliva secretion. The mild infiltration observed in the pancreatic islets of CCR7-deficient mice may also explain why these animals do not develop diabetes spontaneously. The most evident pathogenic manifestation of autoimmunity occurring spontaneously in CCR7-deficient animals is reflected by an ongoing renal dysfunction, as demonstrated by increased levels of urine proteins and decreased creatinine clearance. However, renal dysfunction did not cause severe kidney failure as CCR7-deficient mice displayed an apparently normal lifespan. This is not surprising because the development of fatal renal failure in SLE prone mice results from the interaction of several genetic loci, which cannot by themselves induce severe disease 31, 32. Nevertheless, data presented here demonstrate that the predisposition of CCR7–/– mice for the development of autoimmune disease can be converted into overt disease as exemplified by the development of diabetes following STZ injection.

In conclusion, we have demonstrated that lack of CCR7 signaling renders mice susceptible to the development of multi-organ autoimmunity as demonstrated by the presence of lymphocyte infiltrates in several peripheral organs, increased titers of autoantibodies, increased susceptibility to induced autoimmune diabetes and development of chronic renal disease. Therefore, our study provides new insights into the mechanisms responsible for the initiation of autoimmunity and identifies CCR7-deficiency as a genetic susceptibility factor that contributes to the development of autoimmune diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

The generation of CCR7–/– mice has been described elsewhere 4. CCR7-deficient mice were backcrossed for at least seven generations to the C57BL/6 genetic background. The animals were maintained either under SPF or under germ-free conditions and used at the age of 6 to 26 weeks as indicated in the figure legends. For the majority of the experiments wild-type C57BL/6 mice were bred and maintained in the same animal facility. Remaining control mice were obtained from Charles River (Sulzfeld, Germany), as indicated in the text. All animal experiments have been performed in accordance with institutional guidelines and have been approved by the local committees.

Histology

Following CO2 inhalation, the organs were excised, embedded in OCT compound and frozen on dry ice. The 8-µm thin sections were prepared, air dried and fixed for 15 min in ice-cold acetone. Cryosections were either stained with hematoxylin and eosin or used for immunofluorescence. For immunostaining, sections were blocked with 10% goat or rat serum and stained with antibodies against the indicated markers. The following mAb and reagents were used: homemade anti-CD3 (17A2), anti-CD8ß (RmCD8–2), anti-CD4 (RmCD4–2) and anti-B220 (TIB 146); anti-CD11/c (HL3) and anti-PNAd (Meca79) (BD Bioscience); anti-rat IgG-FITC and anti-Mouse IgG-FITC (Jackson ImmunoResearch) and Alexa Fluor®555 Phalloidin (Molecular Probes).

Enzyme linked immunosorbent assay

Autoantibody levels were detected by ELISA. Briefly, IgG in serial dilutions of sera was captured on 96-well plates (Maxisorb, Nunc) coated with either lysates of the stomach or submandibular gland of wild-type mice. For the preparation of lysates organs of several wild-type animals were pooled and minced in a lyses buffer containing 100 mM KCl, 30 mM NaCl, 35 mM MgCl2, 100 mM Pipes, 12.5 mM EGTA (all Merck), 0.1 mg/mL leupeptin, 0.1 mg pepstatin, 0.1 mM PMSF in isopropanol (all Sigma-Aldrich) and 0.5% NP-40 (Fluka), pH 6.8. Organ lysates were then centrifuged at 1000 × g at 4°C and the supernatants were used for coating the ELISA plates. For the detection of anti-insulin antibodies, plates were coated with insulin (100 ng/well; Sigma-Aldrich). Alpha-fodrin-coated plates (Aesku. Diagnostics) were used to determine alpha-fodrin antibody titers. Detection of dsDNA was performed coating the plates with 10 µg/well Salmon sperm DNA as described elsewhere 33. Bound IgG was measured using peroxidase-conjugated goat anti-mouse IgG (Jackson) and detected with tetramethylbenzidine.

Measurement of saliva secretion

The Saliva secretion test was performed as described elsewhere 29. Briefly, mice were anesthetized with avertin. Pilocarpine hydrochloride (Sigma) was dissolved in double-distilled H2O and injected intraperitoneally at 0.5 µg/g body weight to stimulate saliva production. Saliva was collected with a 100-µL sized pipette immediately after pilocarpine injection for a duration of 10 min. Saliva secretion volumes were normalized to body weight.

Induction and evaluation of autoimmune diabetes

Diabetes induction was performed as described 21. Briefly, age-matched wild-type and CCR7-deficient males were injected intraperitoneally with 40 µg/g body weight STZ daily for 4 consecutive days. Blood glucose levels were monitored twice a week after a period of 6 h fasting using an Accu-Check compact (Roche). Mice were considered diabetic when fasting blood glucose levels exceeded 11.1 mmol/L in two consecutive tests.

Determination of protein and creatinine concentrations in urine

Urine samples of wild-type and CCR7-deficient age-matched mice were collected at two different time points and analyzed independently. Spot urinary protein concentrations were determined using a Bio-Rad Protein-Assay kit. Creatinine concentration was measured by a kinetic colorimetric method using a creatinine assay kit (QuantiChromTM Creatinine assay). Both assays were performed according to the manufacturer's instructions.

Statistical analysis

Statistical analysis was undertaken using GraphPad Prism 3 (GraphPad Software, San Diego, CA). With exception of the data shown in Fig. 2I, data have been analyzed using unpaired t-test. Data shown in Fig. 2I have been subjected to one-way ANOVA with Bonferroni post test. The p values less than or equal to 0.05 were considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We thank Oliver Pabst for valuable suggestions on the manuscript and Thorsten Witte for providing alpha-fodrin coated plates. This work has been supported by a DFG grant (SFB587-B3) to R. Förster. The authors have no competing financial interests.

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