Sara A. Michie

PPAR-δ senses and orchestrates clearance of apoptotic cells to promote tolerance

Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and the silent disposal of apoptotic cells is unknown. Here we show that peroxisome proliferator–activated receptor-δ (PPAR-δ) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-δ decreases expression of opsonins such as complement component-1qb (C1qb), resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific Ppard−/− mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-δ has a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.

Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium.

In the nonobese diabetic (NOD) mouse, lymphocytic and monocytic infiltration of the pancreatic islets leads to beta cell destruction. To investigate the mechanisms by which lymphocytes enter the NOD pancreas, pancreata were immunostained using monoclonal antibodies to a variety of adhesion molecules known to be involved in lymphocyte binding to vascular endothelium, an initial step in the migration of lymphocytes from blood into organized lymphoid and inflamed tissues. These adhesion molecules include: lymphocyte homing receptors involved in tissue-selective binding of lymphocytes to peripheral lymph node (L-selectin) or mucosal lymphoid tissue (LPAM-1, alpha 4 beta 7-integrin) high-endothelial venules (HEV); and HEV ligands peripheral vascular addressin (PNAd) and mucosal vascular addressin (MAdCAM-1). In NOD pancreata, alpha 4 beta 7 is expressed on most infiltrating cells at all stages of insulitis, whereas L-selectin expression is more pronounced on cells in the islets at later stages. During the development of insulitis, MAdCAM-1 and to a lesser extent PNAd became detectable on vascular endothelium adjacent to and within the inflamed islets. The Stamper-Woodruff in vitro assay was used to examine lymphoid cell binding to such vessels. These functional assays show that both the mucosal (MAdCAM-1/alpha 4 beta 7) and the peripheral (PNAd/L-selectin) recognition systems are involved in this binding. Our findings demonstrate that expression of peripheral and mucosal vascular addressins is induced on endothelium in inflamed islets in NOD pancreas, and that these addressins participate in binding lymphoid cells via their homing receptors. This suggests that these adhesion molecules play a role in the pathogenesis of diabetes in these mice by being involved in the migration of lymphocytes from blood into the inflamed pancreas.

Lymphocyte Homing to Bronchus-associated Lymphoid Tissue (BALT) Is Mediated by L-selectin/PNAd, α4β1 Integrin/VCAM-1, and LFA-1 Adhesion Pathways

Bronchus-associated lymphoid tissue (BALT) participates in airway immune responses. However, little is known about the lymphocyte–endothelial adhesion cascades that recruit lymphocytes from blood into BALT. We show that high endothelial venules (HEVs) in BALT express substantial levels of VCAM-1, in marked contrast to HEVs in other secondary lymphoid tissues. BALT HEVs also express the L-selectin ligand PNAd. Anti–L-selectin, anti-PNAd, and anti–LFA-1 mAbs almost completely block the homing of B and T lymphocytes into BALT, whereas anti–α4 integrin and anti–VCAM-1 mAbs inhibit homing by nearly 40%. α4β7 integrin and MAdCAM-1 are not involved. Importantly, we found that mAbs against α4 integrin and VCAM-1 significantly block the migration of total T cells (80% memory phenotype) but not naive T and B cells to BALT. These results suggest that an adhesion cascade, which includes L-selectin/PNAd, α4β1 integrin/VCAM-1, and LFA-1, targets specific lymphocyte subsets to BALT. This high level of involvement of α4β1 integrin/VCAM-1 is unique among secondary lymphoid tissues, and may help unify lymphocyte migration pathways and immune responses in BALT and other bronchopulmonary tissues.

Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression

Keratin polypeptides 8 and 18 (K8/18) are the major intermediate filament proteins of simple-type epithelia. K18 Ser-33 phosphorylation regulates its binding to 14-3-3 proteins during mitosis. We studied the significance of keratin binding to 14-3-3 in transgenic mice that overexpress wild-type or Ser-33→Ala (S33A) K18. In S33A but not wild-type K18-overexpressing mice, pancreatic acinar cell keratin filaments retracted from the basal nuclear region and became apically concentrated. In contrast, K18 S33A had a minimal effect on hepatocyte keratin filament organization. Partial hepatectomy of K18-S33A-overexpressing mice did not affect liver regeneration but caused limited mitotic arrest, accumulation of abnormal mitotic figures, dramatic fragmentation of hepatocyte keratin filaments, with retention of a speckled 14-3-3ζ mitotic cell nuclear-staining pattern that usually becomes diffuse during mitosis. Hence, K18 Ser-33 phosphorylation regulates keratin filament organization in simple-type epithelia in vivo. Keratin binding to 14-3-3 may partially modulate hepatocyte mitotic progression, in association with nuclear redistribution of 14-3-3 proteins during mitosis.

Interferon-α initiates type 1 diabetes in nonobese diabetic mice

With the goal of identifying changes in gene expression in CD4+ T cells during the development of diabetes in the nonobese diabetic (NOD) mouse, we used DNA microarrays to analyze gene expression in CD4+ T cells from the pancreatic draining lymph nodes of NOD/BDC 2.5 T cell receptor transgenic and WT NOD mice at different ages. At 4 and 6 weeks of age, we found up-regulation of a number of genes that are known to be induced by IFN-α. IFN-α levels and IFN-α–producing plasmacytoid dendritic cells were increased in the PLNs of 3- to 4-week-old NOD mice. Moreover, blockade of IFN-α receptor 1 in NOD mice by a neutralizing antibody at 2–3 weeks of age significantly delayed the onset and decreased the incidence of type 1 diabetes, increased the relative number of immature dendritic cells in the PLNs, and enhanced the ability of spleen CD4+ T cells to produce IL-4 and IL-10. These findings demonstrate that IFN-α in the PLNs is an essential initiator in the pathogenesis of type 1 diabetes in NOD mice.

Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant.

Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.

Involvement of β7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the development of diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice develop autoimmune-mediated lymphocytic inflammation of pancreatic islets (insulitis) that leads to β-cell destruction and development of diabetes. Inflamed islets show expression of lymphocyte α4β7 integrin and endothelial mucosal addressin cell adhesion molecule-1 (MAdCAM-1), adhesion molecules involved in tissue-selective migration of lymphocytes to mucosal lymphoid tissues. To elucidate the roles of the mucosal lymphocyte/endothelial adhesion system in the development of diabetes, we treated NOD mice with monoclonal antibody against β7 integrin or MAdCAM-1. Treatment of mice from age 7 to 28 days or 8 to 12 weeks with either antibody led to significant and long-standing protection against the spontaneous development of diabetes and insulitis. In contrast, neither treatment prevented the development of salivary gland inflammation (sialadenitis), indicating that the effect was tissue-selective. Monoclonal antibody treatment had no demonstrable effect on numbers or phenotypes of peripheral lymphocytes or on the immune response to pancreatic islet or exogenous antigens. These data indicate that lymphocyte and endothelial adhesion molecules involved in the migration of lymphocytes into mucosal lymphoid tissues play a role in the development of diabetes in NOD mice. Moreover, the results suggest that treatment of humans with antibodies against tissue-selective lymphocyte or endothelial adhesion molecules may selectively inhibit the development of autoimmune diseases such as diabetes.

The "syncytial variant" of nodular sclerosing Hodgkin's disease

The histologic and immunologic features of an unusual morphologic expression of nodular sclerosing Hodgkins disease, which has been termed the “syncytial variant,” are described. In biopsy material from 18 cases, numerous Reed-Sternberg cell variants were observed in sheets and cohesive clusters, and at least focal evidence of nodular sclerosis was present in each case. The granulocyte antibody anti-Leu Ml reacted with antigenic determinants in Reed-Sternberg cells and atypical variants thereof in 13 of the 18 cases; the lack of staining with antibodies reactive with the leukocyte common (T200) antigen (PD7/26), keratin (AE1), and SI00 protein (polyclonal anti-SlOO) was helpful in excluding non-Hodgkins lymphoma, carcinoma, and melanoma, respectively. This unusual form of nodular sclerosing Hodgkins disease is important to recognize, since it may simulate metastatic neoplasms, thymoma, and non-Hodgkins lymphoma.

Expression of bcl‐2 protein and Ki‐67 nuclear proliferation antigen in benign and malignant cutaneous T‐cell infiltrates

The bcl‐2 protein prolongs cell life by inhibiting apoptosis. Its expression has been studied in a variety of normal tissues and lymphomas but there is minimal information available concerning bcl‐2 expression by benign and malignant cutaneous T‐cells. Therefore, we investigated bcl‐2 expression in a wide variety of cutaneous T‐cell infiltrates using one‐ and two‐color immunohistologic techniques, bcl‐2 was expressed by the majority of lesional CD3+ T‐cells in most cases. This included 22Departments of 26 cases of mycosis fungoides (MF), 3/3 cases of non‐MF cutaneous T‐cell lymphoma, 5/5 cases of lymphomatoid papulosis, 4/4 cases of T‐cell rich cutaneous lymphoid hyperplasia, 2/3 cases of bullous pemphigoid, 2/2 cases of discoid lupus erythematosus and 1/1 case of lichen planus. Titration experiments and comparative studies of tonsil section positive controls revealed that, relative to mantle zone B‐cells, there was over‐ expression of bcl‐2 by a variable subset of T‐cells in most cases. Assessment of multiple biopsies in a subset of MF cases showed stable expression of bcl‐2 over intervals of up to two years. In contrast to the widespread expression of bcl‐2 in both early and advanced MF skin lesions, abundant expression of the nuclear proliferation antigen, Ki‐67, was skewed toward advanced MF skin lesions. Ten percnt or more Ki‐67+ cells were present in 5% of patients with patches/thin plaques, 38% with moderate plaques, 64% with thick plaques and 100% with tumor nodules. Two‐color immunohistologic analysis combined with molecular biologic analysis of clonality in the cases of T‐cell rich cutaneous lymphoid hyperplasia indicated that bcl‐2 expression was both a polyclonal and multi‐lineage phenomenon, suggesting that it occurred by a physiologic rather than mutational mechanism. We conclude that bcl‐2 expression is a common feature of cutaneous T‐cell infiltrates that has minimal differential diagnostic value for distinguishing lymphomas from reactive T‐cell infiltrates. In early MF lesions, abundant expression of bcl‐2 and sparse expression of Ki‐67 suggested that the accumulation of tumor cells during the initial progression of MF may be facilitated by prolonged clonal survival in conjunction with low‐grade clonal proliferation.

Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor β

Tumor-associated macrophages (TAMs) are frequently found in glioblastomas and a high degree of macrophage infiltration is associated with a poor prognosis for glioblastoma patients. However, it is unclear whether TAMs in glioblastomas promote tumor growth. In this study, we found that folate receptor β (FRβ) was expressed on macrophages in human glioblastomas and a rat C6 glioma implanted subcutaneously in nude mice. To target FRβ-expressing TAMs, we produced a recombinant immunotoxin consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRβ monoclonal antibody and Pseudomonas exotoxin A. Injection of the immunotoxin into C6 glioma xenografts in nude mice significantly depleted TAMs and reduced tumor growth. The immunotoxin targeting FRβ-expressing macrophages will provide a therapeutic tool for human glioblastomas.