GM-CSF–deficient Mice Develop SLE.
While earlier work delineated a pulmonary alveolar proteinosis-like disease in GM-CSF– and GM-CSF/IL-3–deficient mice (7
), pathologic analysis of aged mutant animals (8+ months) on the C57Bl/6, but not Balb/c background, unexpectedly revealed immune-mediated glomerulonephritis with B and T cell aggregates in the renal pelvis and liver ( ; similar findings in 22/22 GM-CSF– and 17/17 GM-CSF/IL-3–deficient mice). Consistent with these lesions, a significant proportion of GM-CSF–and GM-CSF/IL-3–deficient mice generated anti-double-stranded DNA antibodies and anti-C1q-reactivity (). Together, these pathologic and serologic features characterize a SLE-like disorder in older mutant animals.
Figure 1. Aged GM-CSF– and GM-CSF/IL-3–deficient mice develop a SLE-like disorder. (A) Membrano-proliferative glomerulonephritis with Ig deposition in a GM-CSF-deficient mouse, anti-κ antibody, original magnification ×400. (B) B220 (more ...)
The mechanisms underlying SLE in several murine models involve the impaired phagocytosis of apoptotic cells (16
). To test whether GM-CSF/IL-3 deficiency results in a similar defect, we injected dexamethasone-induced apoptotic thymocytes into peritoneal cavities and measured their phagocytosis by resident macrophages (13
). Cytospins of peritoneal washings disclosed a marked reduction in the uptake of apoptotic bodies by macrophages from mutant mice compared with wild-type animals () . Similar defects were also present in GM-CSF deficient mice (5/5 animals examined, unpublished data). Moreover, fluorescent labeling of thymocytes with 5- (and 6-) carboxytetramethylrhodamine, succinimidyl ester (5[6
]-TAMRA, SE) revealed that fewer than one-third of the Mac-1–positive cells from GM-CSF/IL-3–deficient mice were associated with apoptotic material, in contrast to more than three-quarters of the Mac-1 positive cells from control animals (). This compromised phagocytosis of apoptotic cells is consistent with prior work showing the defective uptake of pulmonary surfactant, bacteria, and latex beads by alveolar macrophages from mutant mice (17
). Whereas all mutant mice analyzed showed the impaired phagocytosis of apoptotic cells, the variable titers of anti-dsDNA antibodies indicate that additional factors contribute to disease severity.
Figure 2. Impaired phagocytosis of apoptotic cells in GM-CSF– and GM-CSF/IL-3–deficient mice. Dexamethasone-induced apoptotic thymocytes from GM-CSF–deficient mice were injected into the peritoneal cavities of wild-type or GM-CSF/IL-3–deficient (more ...)
GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient Mice.
Despite the autoimmune disease, GM-CSF– and GM-CSF/IL-3-deficient mice failed to manifest an increase in spontaneous tumor formation over 14 mo of observation. As IFN-γ–deficient mice display an enhanced susceptibility to chemical carcinogenesis (18
), we tested whether these cytokines cooperate in tumor suppression by generating compound knockout animals. Surprisingly, GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient mice demonstrated reduced survival compared with parental strains ( A). Whereas the litter sizes were nearly normal, ~40% of the newborn animals succumbed to overwhelming pneumonia during the first few weeks of life ( C). Lung washings consistently grew Pasteurella pneumotropica
, a gram-negative coccobacillus that normally colonizes the oropharynx of rodents without causing disease (21
). Lethal infection in the mutant animals likely reflects, at least in part, diminished bacterial ingestion and killing.
Figure 3. Infection, inflammation, and cancer in GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient mice. (A) Survival curves for mice singly or multiply deficient in GM-CSF, IL-3, and IFN-γ. The numbers of animals per cohort (more ...)
Surviving 2–4-mo-old GM-CSF/IFN-γ–deficient mice manifested no alterations in the numbers of circulating blood cells, but GM-CSF/IL-3/IFN-γ–deficient animals showed a mild eosinophilia (0.29 ± 0.12 × 103
, GM-CSF/IL-3/IFN-γ–deficient versus 0.08 ± 0.03 × 103
, wild-type; P = 0.0001) that was comparable to GM-CSF/IL-3–deficient mice (0.32 ± 0.10 × 103
; reference 9
). The thymi of GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient animals maintained normal T cell maturation, and the spleens contained normal numbers of B220, CD3, CD4, CD8, NK1.1, NK1.1-T, and CD11c-positive cells, although Gr-1 and Mac-1 positive cells were increased twofold.
Over time GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient mice gradually became moribund, with most requiring sacrifice by 14 mo. At autopsy, acute and chronic inflammatory reactions were present in many organs, particularly the lungs, soft tissues, lymph nodes, ovaries, adrenal glands, and liver ( D). Pasteurella pneumotropica and enterococcal species were frequently cultured from these lesions. Bone marrow myeloid hyperplasia and splenic extra-medullary hematopoiesis were associated with the persistent infections. Inflammatory lesions were present in 39/45 mice examined.
In contrast to the intense inflammatory responses, the SLE-like disorder was attenuated in GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient mice compared with GM-CSF– and GM-CSF/IL-3–deficient animals. Lymphoid aggregates were absent from the renal pelvis, and the immune-mediated glomerulonephritis was less severe. Moreover, anti-double-stranded DNA antibodies and anti-C1q reactivity were reduced (). Together, these results reveal a critical role for IFN-γ in the SLE induced by GM-CSF deficiency and are in accordance with the requirement for IFN-γ in the SLE caused by defective fas/fas-ligand function (22
Within the background of chronic infection and inflammation, a high proportion of GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient mice developed lymphoproliferative diseases ( B, online supplemental Table S1). Atypical lymphoid hyperplasias (23
) originating in mesenteric lymph nodes or Peyer's patches frequently evolved to mature B cell lymphomas (B220+
by immunohistochemistry) involving the liver, spleen, and other organs (36/40 lymphomas examined were B cell). Pathologically, the tumors ranged from low-grade lesions with plasmacytoid features to high-grade large cell expansions with abundant mitoses (). Southern analysis of immunoglobulin light and heavy chain gene rearrangements established that the proliferations were clonal ( A, and online supplemental Fig. S1 A). Spectral karyotyping further disclosed chromosomal translocations and aneuploidy in 3 of 6 tumors examined ( B, and online Supplemental Fig. S1 B). Three of three lymphomas tested were efficiently transplanted into young GM-CSF/IL-3/IFN-γ–deficient recipients (unpublished data).
Figure 4. B cell lymphomas. (A) Tumor-derived DNA from six different lymphomas was analyzed by southern using a Jκ probe. The germline band is 2.7 kb. (B) Single cell lymphoma suspensions were stimulated with anti-CD40 antibodies, and spectral karyotyping (more ...)
In addition to the lymphomas, nearly 50% of the GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient mice developed solid tumors ( B, online supplemental Table S1, online supplemental Table S2). The pathologies observed ranged from lesions that resemble well-described benign tumors in the human to carcinomas with metastases. Ovarian tumors were the most common, with 6/13 double deficient and 4/4 triple deficient females manifesting choriocarcinomas ( G), luteomas, or teratomas. The broad spectrum of tumors also included carcinomas of the biliary tract/liver ( H), salivary gland, and bladder as well as adenomas/hyperplasias of the pancreatic islets, seminal vesicle, and osteochondral junction. It is noteworthy that 4/29 double deficient and 4/16 triple deficient mice showed multiple solid lesions.
Tumor development required the combined deficiency of GM-CSF and IFN-γ. Histopathologic analysis of aged animals (12–17 mo) revealed no tumors in 7 wild-type mice, 2 cases of atypical hyperplasia and 1 luteoma in 15 GM-CSF-deficient mice, 2 cases of atypical hyperplasia in 17 GM-CSF/IL-3–deficient mice, and 1 luteoma and 1 islet cell hyperplasia in 13 IFN-γ–deficient mice. Moreover, gross autopsy of 28 additional IFN-γ–deficient animals up to 27 mo of age disclosed only a single case of lymphoma (B220+). Most GM-CSF- and GM-CSF/IL-3–deficient mice succumbed to pulmonary alveolar proteinosis by 18 mo, precluding a longer-term study of spontaneous tumor development in these strains.
To explore the mechanisms underlying spontaneous tumor formation in the compound cytokine deficient mice, we characterized potential precursor lesions in B cell lymphomagenesis. Although secondary lymphoid tissues of 2–3 mo old mice did not show evidence of atypical hyperplasia or clonal proliferation, germinal centers were enlarged. Consistent with these findings, serum IgG1 levels were higher in mutant mice than controls (1,811 ± 329, GM-CSF/IFN-γ–deficient versus 921 ± 172, wild-type; P = 0.0003); serum IgG2b (4,431 ± 2,150 versus 2,240 ± 730) and IgE levels (845 ± 709 versus 330 ± 427) were also increased, but these did not reach statistical significance.
Unresolved infection and inflammation likely contribute to the persistent B cell activation in GM-CSF/IFN-γ– and GM-CSF/IL-3/IFN-γ–deficient mice. To determine whether intrinsic B cell defects were also involved, we evaluated the responses of purified B cells to anti-CD40 antibodies or lipopolysaccharide. No significant differences in the cell cycle profiles or up-regulation of surface MHC II, B7–1, and fas expression were detected between the mutant and wild type cells after 48 h of stimulation (unpublished data). Nonetheless, activated B cells from GM-CSF/IL-3/IFN-γ–deficient mice manifested a resistance to fas-mediated apoptosis (P < 0.0001, versus wild-type), whereas activated B cells from IFN-γ–, GM-CSF–, or GM-CSF/IL-3–deficient animals displayed fas sensitivities equivalent to controls ( C). The killing induced with etoposide, staurosporine, and actinomycin, in contrast, was comparable among all the strains.
As soluble factors modulate target cell fas sensitivity (24
), we tested whether conditioned media from activated wild-type B cells could reverse the defective response of GM-CSF/IL-3/IFN-γ–deficient B cells. As illustrated in D, the addition of wild-type supernatants to mutant cultures restored the fas sensitivity, suggesting that the impaired function of one or more cytokines might be responsible for the defect. Indeed, anti–TNF-α antibodies significantly inhibited fas killing in wild-type B cells, and supernatants from wild-type cultures contained fivefold more immunoreactive TNF-α (6.7 ± 0.44 pg/5 × 106
cells) than GM-CSF/IL-3/IFN-γ–deficient cultures (1.3 ± 0.21 pg/5 × 106
cells), as measured by ELISA. The restoration of normal TNF-α levels in the mutant cultures with recombinant cytokine partially corrected the fas resistance ( D). However, the restoration of both wild-type TNF-α and IFN-γ levels (2.5 ± 0.06 pg/5 × 106
cells) completely restored the fas sensitivity of mutant cells. These results establish a dual requirement for TNF-α and IFN-γ in fas-mediated B cell apoptosis. As aged fas-deficient mice harbor B cell lymphomas (25
), the apoptotic defect delineated here may contribute to tumor development. However, further studies are required to determine whether a similar TNF-α deficiency is present in vivo.
Antibiotics Suppress Tumor Formation.
As microbial agents contribute to lymphomagenesis and solid tumor development in humans, we maintained a cohort of 24 GM-CSF/IL-3/IFN-γ-deficient mice from birth on enrofloxacin. Consistent with the ability of this antibiotic to suppress Pasteurella pneumotropica
and enterococcal species (21
), early infectious deaths were eliminated from the cohort. Moreover, antimicrobial therapy prevented tumor formation in aged animals. No lymphomas or solid tumors were detected in 19 mice autopsied at 14 mo (P < 0.001 for lymphomas, P < 0.001 for all solid tumors, and P = 0.03 for carcinomas, by the Fisher exact test, compared with the untreated cohort). One animal showed atypical thymic hyperplasia; 3 mice died of fighting-related injuries and 2 of unknown causes between 8 and 12 mo. The suppression of tumor formation was associated with a marked reduction in chronic inflammatory lesions; of 9 animals studied by histopathology, only 1 showed mild cholangitis and pancreatitis. In contrast, CD40-activated B cells from enrofloxacin-treated mice displayed a comparable fas-resistance to lymphocytes from untreated GM-CSF/IL-3/IFN-γ–deficient animals (2 mice analyzed, unpublished data). Additional experiments are required to determine whether anti-microbial therapy will suppress tumor formation over a longer time period.