Michelle Krakowski

Inflammatory cytokines in the brain: Does the CNS shape immune responses?

Immune responses in the central nervous system (CNS) have traditionally been regarded as representing the intrusion of an unruly, ill-behaved mob of leukocytes into the well-ordered and organized domain of thought and reason. However, results accumulated over the past few years suggest that, far from being an immunologically privileged organ, T lymphocytes may be regular and frequent visitors to the CNS, for purposes of immune surveillance. Here, Trevor Owens and colleagues propose that the brain itself can regulate or shape immune responses therein. Furthermore, given that the immune cells may be subverted to autoimmunity, they suggest that the study of inflammatory autoimmune disease in the brain may shed light on the ability of the local environment to regulate immune responses.

Pancreatic Expression of Keratinocyte Growth Factor Leads to Differentiation of Islet Hepatocytes and Proliferation of Duct Cells

Keratinocyte growth factor, (KGF), a member of the fibroblast growth factor (FGF) family, is involved in wound healing. It also promotes the differentiation of many epithelial tissues and proliferation of epithelial cells as well as pancreatic duct cells. Additionally, many members of the highly homologous FGF family (including KGF), influence both growth and cellular morphology in the developing embryo. We have previously observed elevated levels of KGF in our interferon-gamma transgenic mouse model of pancreatic regeneration. To understand the role of KGF in pancreatic differentiation, we generated insulin promoter-regulated KGF transgenic mice. Remarkably, we have found that ectopic KGF expression resulted in the emergence of hepatocytes within the islets of Langerhans in the pancreas. Additionally, significant intra-islet duct cell proliferation in the pancreata of transgenic KGF mice was observed. The unexpected appearance of hepatocytes and proliferation of intra-islet duct cells in the pancreata of these mice evidently stemmed directly from local exposure to KGF.

Glutamate metabolism is down-regulated in astrocytes during experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) was induced in SJL/J mice by adoptive transfer of MBP‐reactive T cells in order to investigate the role of astrocytes in pathology. GFAP protein and mRNA expression (analyzed using semi‐quantitative Western blot and RT‐PCR techniques) were upregulated in the spinal cord of mice, which had developed a complete paralysis of hind‐ and fore‐limbs and tail (grade 4 EAE), thus establishing that reactive gliosis occurred under these experimental conditions. Within the same samples and using similar techniques, we found that glutamine synthetase (GS) and glutamate dehydrogenase (GDH) expression were dramatically reduced. These two astrocytic enzymes are responsible for degradation of glutamate, the most abundant excitatory neurotransmitter in the brain. Since elevated levels of glutamate may be neurotoxic, we propose that the decreased capacity of astrocytes to metabolize glutamate may contribute to EAE pathology. GLIA 20:79–85, 1997.

Interferon-γ in Progression to Chronic Demyelination and Neurological Deficit Following Acute EAE☆

The cytokine interferon-gamma (IFNgamma) is implicated in the induction of acute CNS inflammation, but it is less clear what role if any IFNgamma plays in progression to chronic demyelination and neurological deficit. To address this issue, we have expressed IFNgamma in myelinating oligodendrocytes of transgenic mice. MHC I immunostaining and iNOS mRNA were upregulated in their CNS, but such transgenic mice showed no spontaneous CNS inflammation or demyelination, and the incidence, severity, and histopathology of experimental autoimmune encephalomyelitis (EAE) were similar to nontransgenic controls. In contrast to control mice, which remit from EAE with resolution of glial reactivity and leukocytic infiltration, transgenics showed chronic neurological deficits. While activated microglia/macrophages persisted in demyelinating lesions for over 100 days, CD4(+) T lymphocytes were no longer present in CNS. IFNgamma therefore may play a role in chronic demyelination and long-term disability following the induction of demyelinating disease. Because IFNgamma may have neural as well as immune-infiltrating origins, these findings generate a new perspective on its role in the CNS.

Chemokine expression in GKO mice (lacking interferon-gamma) with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) considered to be an animal model for multiple sclerosis (MS). The detailed mechanism that specifies accumulation of inflammatory cells within the CNS in these conditions remains a subject of active investigation. Chemokines including IP-10, GRO-alpha, MCP-1 are produced in EAE tissues selectively by parenchymal astrocytes, but the regulatory stimuli that govern this expression remain undetermined. The unexpected occurrence of increased EAE susceptibility in Balb/c GKO mice (lacking IFN-gamma) offered an opportunity to examine the spectrum of chemokine expression during immune-mediated inflammation in the absence of a single regulatory cytokine. We found that chemokines MCP-1 and GRO-alpha were upregulated in the CNS of mice with EAE despite the GKO genotype. IP-10, which is highly expressed in the CNS of mice with an intact IFN-gamma gene and EAE, was strikingly absent. In vitro experiments confirmed that IFNgamma selectively stimulates astrocytes for IP-10 expression. These results indicate that IP-10 is dependent upon IFN-gamma for its upregulation during this model disease, and document directly that astrocyte expression of chemokines during EAE is governed by pro-inflammatory cytokines.

Transcription factor expression during pancreatic islet regeneration.

Recent studies by a number of laboratories have identified transcription factors that are involved in pancreatic development. Indeed, marked abnormalities in pancreatic development result from deficiencies in these molecules, which include, among others, PDX-1, islet-1 (Isl-1), and Pax-6. These studies have prompted us to evaluate the expression of Isl-1 and Pax-6 in the pancreas of the interferon-gamma (IFNgamma) transgenic mouse, which exhibits new islet growth and expansion of ducts throughout the life of the animal. We have previously demonstrated that PDX-1 is strikingly expressed in the ducts of the IFNgamma transgenic mouse. This latter observation compelled us to examine expression of hepatocyte nuclear factor-3beta (HNF3beta), which mediates PDX-1 gene transcription, in the IFNgamma transgenic pancreas as well. As a result of these studies, we now demonstrate marked expression of these transcription factors in the pancreatic ducts of IFNgamma transgenic mice. These data suggest a role for these transcription factors during pancreatic regeneration in the IFNgamma transgenic mouse.

Granulocyte macrophage-colony stimulating factor (GM-CSF) recruits immune cells to the pancreas and delays STZ-induced diabetes

Granulocyte macrophage‐colony stimulating factor (GM‐CSF) is one of the most widely used growth factors for enhancing immune responses and is known to recruit and activate antigen‐presenting cells (APCs). This study hypothesized that overexpression of this cytokine within the pancreatic β‐cells would recruit, expand, and activate APCs. The question was whether this would lead to tolerance or autoimmunity to pancreatic antigens. This possibility was tested by preparing transgenic mice (ins‐GM‐CSF) whose islets expressed murine GM‐CSF. By 6–8 weeks of age, these mice developed a profound mononuclear cell infiltration that often overwhelmed the exocrine pancreas, although no changes in enzyme or hormone function were apparent. The majority of the mononuclear infiltrate within the pancreas was identified as F4/80+ macrophages. Transgenic ins‐GM‐CSF mice had splenomegaly due to a massive increase in the macrophage population. Additionally, mononuclear cells were found within the livers of transgenic mice, with F4/80+ cells also identified within the infiltrate, indicating that GM‐CSF‐activated mononuclear cells circulated to organs other than the pancreas. To assess the disease potential, this study tested whether macrophage recruitment to the pancreas might accelerate or protect the islets from diabetes. It was found that the induction of diabetes by low‐dose streptozotocin (STZ) was delayed and reduced within ins‐GM‐CSF transgenic mice, in comparison with negative littermates. Together, these data highlight the role of GM‐CSF in recruiting APCs such as macrophages. Advanced cellular infiltration does not overtly harm, and may even protect, pancreatic function, as seen with the delay in chemically induced diabetes. Copyright

Immune cell entry to the CNS — a focus for immunoregulation of EAE

T-cell-derived cytokines are therefore individually unnecessary and collectively insufficient for microglial response. This somewhat provocative interpretation does not exclude a role for T-cell cytokines in induction of a microglial response in EAE, but it may be easier to show a non-requirement then to prove such a role. The point that emerges is that cytokine production in the CNS parenchyma is itself dependent on the prior infiltration of immune cells, and that without immune cell entry, EAE does not occur. This identifies events at the BBB, and in particular in the perivascular space, as critical immunoregulatory events in development and progression of EAE.

Increased islet antigen presentation leads to type-1 diabetes in mice with autoimmune susceptibility

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is frequently used in preclinical and clinical protocols to modulate autoimmune responses, bone marrow transplants, and recovery from immune ablative therapies. The immunological outcome of such therapies is not fully understood. We tested the hypothesis that GM‐CSF would enhance the maturation of antigen‐presenting cells, facilitating presentation of β‐cell autoantigens to autoreactive T cells. We found that islet expression of GM‐CSF greatly enhanced disease in male mice. Islet‐derived APC but not splenic APC showed markedly enhanced capacity to stimulate in vitro proliferative responses of islet‐antigen‐specific autoreactive T cells. In vivo transfer of CD8+ and CD4+ T cells demonstrate that autoreactive T cells undergo extensive division in pancreatic lymph nodes of GM‐CSF‐transgenic mice compared with wild‐type NOD male mice. Together, the results presented here demonstrate that expression of GM‐CSF in the pancreas can enhance autoimmunity in disease‐susceptible mice.

IFN-γ overexpression within the pancreas is not sufficient to rescue Pax4, Pax6, and Pdx-1 mutant mice from death

In the presence of interferon-&ggr; (IFN-&ggr;), pancreatic ductal epithelial cells grow continuously, and islets undergo neogenesis. To determine whether these new islets are derived from conventional precursors, we tested whether IFN-&ggr; can complement the loss of transcription factors known to regulate pancreatic development. We analyzed the effect of a transgene on lethality in mice lacking the transcription factors Pax4, Pax6, or Pdx-1, by intercrossing such mice with transgenic mice whose pancreatic cells make IFN-&ggr; (ins-IFN-&ggr; mice). However, IFN-&ggr; expression did not rescue these mice from the lethal mutations, because no homozygous knockout mice carrying the IFN-&ggr; transgene survived, despite the survival of all other hemizygous gene combinations. This outcome demonstrates that the pathway for IFN-&ggr; regeneration requires the participation of Pax4, Pax6, and Pdx-1. We conclude that the striking islet regeneration observed in the ins-IFN-&ggr; NOD strain is regulated by the same transcription factors that control initial pancreatic development.