Ilona Ormsby

Endogenous Transforming Growth Factor β1 Suppresses Inflammation and Promotes Survival in Adult CNS

Transforming growth factor β1 (TGFβ1) is a pleiotropic cytokine with potent neurotrophic and immunosuppressive properties that is upregulated after injury, but also expressed in the normal nervous system. In the current study, we examined the regulation of TGFβ1 and the effects of TGFβ1 deletion on cellular response in the uninjured adult brain and in the injured and regenerating facial motor nucleus. To avoid lethal autoimmune inflammation within 3 weeks after birth in TGFβ1-deficient mice, this study was performed on a T- and B-cell-deficient RAG2−/− background. Compared with wild-type siblings, homozygous deletion of TGFβ1 resulted in an extensive inflammatory response in otherwise uninjured brain parenchyma. Astrocytes increased in GFAP and CD44 immunoreactivity; microglia showed proliferative activity, expression of phagocytosis-associated markers [αXβ2, B7.2, and MHC1 (major histocompatibility complex type 1)], and reduced branching. Ultrastructural analysis revealed focal blockade of axonal transport, perinodal damming of axonal organelles, focal demyelination, and myelin debris in granule-rich, phagocytic microglia. After facial axotomy, absence of TGFβ1 led to a fourfold increase in neuronal cell death (52 vs 13%), decreased central axonal sprouting, and significant delay in functional recovery. It also interfered with the microglial response, resulting in a diminished expression of early activation markers [ICAM1 (intercellular adhesion molecule 1), α6β1, and αMβ2] and reduced proliferation. In line with axonal and glial findings in the otherwise uninjured CNS, absence of endogenous TGFβ1 also caused an ∼10% reduction in the number of normal motoneurons, pointing to an ongoing and potent trophic role of this anti-inflammatory cytokine in the normal as well as in the injured brain.

TGF-β1 Regulates Lymphocyte Homeostasis by Preventing Activation and Subsequent Apoptosis of Peripheral Lymphocytes

TGF-β1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-β1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-β1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1−/− mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-γ and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1−/− T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1−/− T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4+ T cells have elevated intracellular Ca2+ levels. However, upon subsequent stimulation in vitro, increases in Ca2+ levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1−/− splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca2+ levels.

Strain dependency of TGFβ1 function during embryogenesis

There is incomplete penetrance to Tgfb1 knockout phenotypes. About 50% of Tgfb1 homozygous mutant (Tgfb1−/−) and 25% of Tgfb1 heterozygous (Tgfb1+/−) embryos die during embryogenesis. In a mixed NIH/Ola × C57BL/6J/Ola × 129 background partial embryonic lethality of the Tgfb1−/− embryos occurs due to defective yolk sac vasculopoiesis and/or hematopoiesis. We show here that on a predominantly CF‐1 genetic background, lack of TGFβ1 causes a pre‐morula lethality in about 50% of the null embryos. This partial lethality is not reversed by transfer of Tgfb1−/− embryos to Tgfb1+/+ hosts. The extent of embryonic lethality in Tgfb1−/− embryos ranges in a background dependent manner from 20% to 100%. Based on these and other studies it is clear that TGFβ1 acts at two distinct phases of embryogenesis: pre‐implantation development and yolk sac vasculogenesis/hematopoiesis. The susceptibility for the pre‐implantation lethality depends on a small number of genetic modifiers since a small number of backcrosses onto the high susceptibility strain C57BL/6 leads to complete penetrance of the lethality. Mol. Reprod. Dev. 52:341–349, 1999.

Wound healing in the transforming growth factor‐β1—deficient mouse

To investigate the role of transforming growth factor‐β1 in tissue repair, we performed wound healing studies in the transforming growth factor‐β1—deficient mouse with targeted disruption of the transforming growth factor‐β1 gene. Transforming growth factor‐β1—deficient mice exhibit no obvious developmental defects and are phenotypically normal until approximately 3 weeks of age when a severe wasting syndrome develops, accompanied by an overwhelming inflammatory response resulting in multisystem organ failure and death. Full‐thickness 0.5 × 0.5 cm skin wounds were created on the backs of 10‐day‐old mice (wild type or heterozygous controls versus homozygous transforming growth factor‐β1—deficient mutants) and covered with a nonabsorbent dressing (OpSite). Serial wound measurements were made, and percentage of wound closure over time was determined. On day 10, wounds and liver were harvested for histologic and molecular analysis. Histologic scores were assigned (1 [no healing] to 12 [complete healing]) on the basis of granulation tissue formation, vascularity, collagen deposition, and epithelialization. Reverse transcription—polymerase chain reaction was performed to detect messenger RNA transcripts for transforming growth factor‐β1, transforming growth factor‐β2, platelet‐derived growth factor A‐chain and B‐chain, interleukin‐1β and ‐6, and tumor necrosis factor‐α in unwounded skin, day 10 wounds, and liver. No significant differences in wound closure were observed until day 10. Weight gain, however, was significantly decreased in the mutant animals as early as day 6. Histologic scores were significantly lower in the transforming growth factor‐β1—deficient mutants (5.4 ± 0.6 versus 11.1 ± 0.3, p < 0.01, Wilcoxon rank‐sum test) and showed decreased granulation tissue formation, vascularity, collagen deposition, and epithelialization and a marked inflammatory infiltrate. As expected, transforming growth factor‐β1 was expressed in controls but not mutants. Transforming growth factor‐β2, platelet‐derived growth factor A‐chain and B‐chain, and tumor necrosis factor‐α were constitutively expressed in unwounded skin, day 10 wounds, and liver of both controls and mutants. Interleukin‐1β and ‐6, however, were induced after wounding. Early wound healing in the transforming growth factor‐β1—deficient mouse proceeds relatively normally because of upregulation or functional redundancy of other growth factors or possibly because of maternal rescue by means of transforming growth factor‐β1 transmitted in milk. Loss of transforming growth factor‐β1 regulation ultimately results in a marked inflammatory response, as evidenced by the histologic appearance of the wound and increased expression of the inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β and 6). The severe wasting syndrome (marked by weight loss) undoubtedly has an adverse effect on wound healing.

TGFβ1 Inhibits Ca2+-Calcineurin-Mediated Activation in Thymocytes

TGFβ1 is a polypeptide growth modulatory and differentiation factor involved in many biological processes including immune homeostasis and self-tolerance. Tgfb1 knockout mice die around weaning age due to severe inflammation in most major organ systems, but the mechanism underlying this disease is not understood. In this study we demonstrate that Tgfb1−/− CD4+CD8+ and CD4+CD8− thymocytes are hyperresponsive to receptor-mediated and receptor-independent mitogenic stimulation. A suboptimal concentration of ionomycin in the presence of PMA fully activates Tgfb1−/− thymocytes, whereas the inhibitors of Ca2+ influx and calcineurin, EGTA and FK506, eliminate the hyperresponsiveness. Hence, the hypersensitivity of Tgfb1−/− thymocytes is due to a lowered threshold for Ca2+-dependent activation. Further, we demonstrate that the hypersensitivity of thymocytes results from the absence of TGFβ1 and not from the inflammatory environment because the thymocytes are hyperresponsive in preinflammatory-stage Tgfb1−/− mice. Our results suggest for the first time that TGFβ1 functions to inhibit aberrant T cell expansion by maintaining intracellular calcium concentration levels low enough to prevent a mitogenic response by Ca2+-independent stimulatory pathways alone. Consequently, TGFβ1 prevents autoimmune disease through a Ca2+ regulatory pathway that maintains the activation threshold above that inducible by self-MHC-TCR interactions.

Self-antigen recognition by TGFβ1-deficient T cells causes their activation and systemic inflammation

To investigate whether the multifocal inflammatory disease in TGFβ1-deficient mice is caused by self-antigen (self-Ag)-specific autoreactive T cells, or whether it is caused by antigen independent, spontaneous hyperactivation of T cells, we have generated Tgfb1−/− and Tgfb1−/− Rag1−/− mice expressing the chicken OVA-specific TCR transgene (DO11.10). On a Rag1-sufficient background, Tgfb1−/− DO11.10 mice develop a milder inflammation than do Tgfb1−/− mice, and their T cells display a less activated phenotype. The lower level of activation correlates with the expression of hybrid TCR (transgenic TCRβ and endogenous TCRα), which could recognize self-Ag and undergo activation. In the complete absence of self-Ag recognition (Tgfb1−/− DO11.10 Rag1−/− mice) inflammation and T-cell activation are eliminated, demonstrating that self-Ag recognition is required for the hyper-responsiveness of TGFβ1-deficient T cells. Thus, TGFβ1 is required for the prevention of autoimmune disease through its ability to control the activation of autoreactive T cells to self-Ag.

The morphology and biologic behavior of human glioblastoma growing in nude mice

Seven human glioblastomas (five small cell glioblastomas [SCG] and two anaplastic astrocytomas with giant cells (AAI) grown in serial passage in BALB/c nude mice and nude rats, were studied histologically and compared to human donor tumors. Four SCG maintained many basic features seen in donor tissue, i.e., cell type, tendency to produce micronecrotic palisading (MNP), high cellularity, numerous mitoses. Significant vascular proliferation was seen only in nude rat hosts of one of the SCG lines transplanted from mice at passage 11. Giant capillaries, cyst formation and hemorrhages were features of large (1.0 cm) heterotransplanted SCG. One SCG altered morphology from first passage, showing an adenoid pattern and mucinous change. Both AA preserved original tissue characteristics in initial passages. Later dedifferentiation occurred with small cells predominating. These small cells were larger and rounder than those of the SCG. Large AA showed central necrosis but rarely MNP, hemorrhages and focal inflammatory infiltrates. Cancer 58:1061‐1069, 1986.

Role of Transforming Growth Factor 1 in Lymphocyte Development and Death

INTRODUCTION. Transforming growth factor β1 (TGFβ1) is a polypeptide growth factor known to exert multiple functions during development and in the adult stage as well (1-2). TGFβ1 knockout mice are normal at the time of birth and do not exhibit any developmental defect. After one week of birth these mice start developing multifocal inflammatory lesions and eventually die around three weeks of age (3). Further studies revealed that T lymphocytes are the primary effectors in this phenotype (Doetschman, unpublished observation). During T cell development in the thymus, T cell progenitors undergo massive proliferation and around 95% of them undergo apoptosis. Positively selected CD4CD8 double positive T cells undergo thymic selection where cells that recognize self-antigens with high affinity are induced to undergo apoptosis (negative selection) (4-5). Any perturbations in the thymic education process might lead to export of self-reactive T cells to the periphery. In order to find the role of TGFβ1 in preventing the inflammation, we have studied the lymphocyte apoptosis and proliferation.

STRAIN DEPENDENCY OF TRANSFORMING GROWTH FACTOR-BETA 1 (TGF-betal) REQUIREMENT FOR PREIMPLANTATION MOUSE DEVELOPMENT. |[dagger]| 352

Murine embryos express TGF-betal as early as 2-4 cell stage. To investigate the functions of TGF-betal in vivo, our laboratory disrupted the TGF-betal gene (Shull et al, Nature 1992: 359, 693-99). Genotypes of pups born from intercrosses of animals heterozygous for the disrupted TGF-betal allele in the 129×CF-1 strain revealed a significant deviation from the mendelian ratio of 1:2:1. Of 806 live offsprings born, 280 were homozygous wild type(+/+), 413 were heterozygous (+/-) and 113 were homozygous mutant (-/-), giving a ratio of 1:1.5:0.4. The +/+ data has been normalized to 1. To determine the gestational stage at which embryonic lethality occurred, genotyping of embryos derived from heterozygous crosses were undertaken at various developmental stages. In the 129×CF-1 strain, genotypic analysis of 398 blastocysts showed the ratio to be 1:1.3:0.5 (+/+: +/-: -/-). Next, in vitro development of preimplantation embryos derived from TGF-betal heterozygous crosses in the same strain was assessed. 134 four cell stage embryos out of 140 could be successfully cultured to healthy compacted morula/blastocyst stage embryos, the culturing efficiency being no different from controls. To demonstrate that the embryonic loss cannot be accounted for by differential fertilization rates, crosses were undertaken between +/+ and+/- animals in both sexes. Of 170 pups born to TGF-betal +/- male and +/+ female the ratio of +/+: +/- pups was 1:1.5. When TGF-betal +/+ males were mated with TGF-betal +/- females, the ratio of +/+: +/- pups was 1: 0.82(N=180) (p>0.05). Thus there was no under-representation of +/- embryos at fertilization. These results suggest that in the 129×CF-1 strain, approximately 50% TGF-betal -/- embryos fail to develop past the first two cell divisions in preimplantation embryogenesis.