Katarina Håkansson

FGF-2-Responsive Neural Stem Cell Proliferation Requires CCg, a Novel Autocrine/Paracrine Cofactor

We have purified and characterized a factor, from the conditioned medium of neural stem cell cultures, which is required for fibroblast growth factor 2s (FGF-2) mitogenic activity on neural stem cells. This autocrine/paracrine cofactor is a glycosylated form of cystatin C (CCg), whose N-glycosylation is required for its activity. We further demonstrated that, both in vitro and in vivo, neural stem cells undergoing cell division are immunopositive for cystatin C. Finally, we showed in vivo functional activity of CCg by demonstrating that the combined delivery of FGF-2 and CCg to the adult dentate gyrus stimulated neurogenesis. We propose that the process of neurogenesis is controlled by the cooperation between trophic factors and autocrine/paracrine cofactors, of which CCg is a prototype.

Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene

AIMS: Increased or altered activities of cysteine proteases have been implicated in serious human disorders such as cancer, rheumatoid arthritis, sepsis, and osteoporosis. To improve the current knowledge of the regulatory role of a major mammalian cysteine protease inhibitor, cystatin C, in such disease processes, a cystatin C deficient mouse was generated and characterized. METHODS: The mouse cystatin C gene was inactivated by insertion of a bacterial neo gene through homologous recombination in 129/Sv embryonic stem cells. Embryonic stem cell clones were injected into C57BL/6J blastocysts followed by injection of the blastocysts into pseudopregnant female mice. F1 offspring with agouti coat colour after mating of chimaeric males with C57BL/6J females were examined by DNA analysis, and mice carrying the targeted mutation were intercrossed to obtain homozygous cystatin C deficient (CysC-/-) mice. To study the role of cysteine proteases and their inhibitors in metastasis, the spread of B16-F10 melanoma cells in CysC-/- and wild-type mice was compared. Analysis of the formation of remote metastases was carried out by intravenous injection of beta-galactosidase transfected B16-F10 cells and subsequent determination of cancer cell colonies in the lungs. RESULTS: Cystatin C deficient mice were fertile and showed no gross pathological abnormality up to 6 months of age. Compared with wild-type mice, seven times fewer large metastatic colonies were counted by means of a dissecting microscope in CysC-/- mice two weeks after tail vein injection of B16-F10 cells. At all of eight time points from 15 minutes to two weeks after intravenous injection of tumour cells, the CysC-/- mice had significantly fewer lung metastases. The observed differences were smaller when beta-galactosidase transfected cells were used to allow counting of small colonies. Subcutaneous and intracerebral tumour growth was not different in the CysC-/- mice. CONCLUSIONS: Cystatin C concentrations in vivo might influence metastasis in some tissues. The decreased metastatic spread of B16-F10 cells in CysC-/- mice is the result of both reduced seeding and reduced growth of tumour cells in their lungs.

Lack of the Cysteine Protease Inhibitor Cystatin C Promotes Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Degradation of extracellular matrix plays an important role in growth and destabilization of atherosclerotic plaques. Cystatin C, inhibitor of the collagen- and elastin-degrading cysteine proteases of the cathepsin family, is produced by virtually all cell types. It is present in the normal artery wall but severely reduced in human atherosclerotic lesions. Methods and Results—To determine the functional role of cystatin C in atherosclerosis, we crossed cystatin C–deficient (cysC−/−) mice with apolipoprotein E–deficient (apoE−/−) mice. After 25 weeks of atherogenic diet, mice lacking apoE and cystatin C (cysC−/− apoE−/−) had larger subvalvular plaques compared with cysC+/+ apoE−/− mice (766 000±20 000 &mgr;m2 per section versus 662 000±19 000 &mgr;m2 per section; P=0.001), suggesting an atheroprotective role of cystatin C. The plaques from cysC−/− apoE−/− mice were characterized by increased total macrophage content. To determine which cellular source is important for the antiatherosclerotic effect of cystatin C, we performed bone marrow transplantations. ApoE−/− mice were transplanted with either cysC−/− apoE−/− or cysC+/+ apoE−/− bone marrow. No significant differences in plaque area, macrophage, collagen, or lipid content of subvalvular lesions between the 2 groups were detected. Conclusions—The result suggests that the protective role of cystatin C in atherosclerosis is dependent primarily on its expression in nonhematopoietic cell types.

Induction of Lectin-like Transcript 1 (LLT1) Protein Cell Surface Expression by Pathogens and Interferon-γ Contributes to Modulate Immune Responses

Background: CD161 expressed by NK cells and T cells interacts with LLT1. Results: LLT1 expression profile reveals LLT1 is induced by pathogens and IFN-γ and LLT1/CD161 interaction inhibits NK cell functions whereas it costimulates T cells. Conclusion: The link between LLT1 expression and pathogen stimulation points toward a role in modulating immune responses to pathogens Significance: LLT1/CD161 interaction is relevant in immunity to infection. CD161 is a C-type lectin-like receptor expressed on human natural killer (NK) cells and subsets of T cells. CD161 has been described as an inhibitory receptor that regulates NK cell-mediated cytotoxicity and IFN-γ production. Its role on T cells has remained unclear. Studies have shown that triggering of CD161 enhances NK T cell proliferation and T cell-IFN-γ production while inhibiting TNF-α production by CD8+ T cells. Lectin-like transcript 1 (LLT1), the ligand of CD161, was found to be expressed on Toll-like receptor (TLR)-activated plasmacytoid and monocyte-derived dendritic cells (DC) and on activated B cells. Using newly developed anti-LLT1 mAbs, we show that LLT1 is not expressed on the surface of circulating B and T lymphocytes, NK cells, monocytes, and dendritic cells but that LLT1 is up-regulated upon activation. Not only TLR-stimulated dendritic cells and B cells but also T cell receptor-activated T cells and activated NK cells up-regulate LLT1. Interestingly, IFN-γ increases LLT1 expression level on antigen-presenting cells. LLT1 is also induced on B cells upon viral infection such as Epstein-Barr virus or HIV infection and in inflamed tonsils. Finally, expression of LLT1 on B cells inhibits NK cell function but costimulates T cell proliferation or IFN-γ production, and coengagement of CD161 with CD3 increases IL-17 secretion. Altogether, our results point toward a role for LLT1/CD161 in modulating immune responses to pathogens.

The Protease Inhibitor Cystatin C Is Differentially Expressed among Dendritic Cell Populations, but Does Not Control Antigen Presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class II (MHC II) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC II chaperone Ii, and hence in the formation of MHC II-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8+ DC, but not CD4+ or CD4−CD8− DC, synthesize CyC, which accumulates in MHC II+Lamp+ compartments. However, Ii processing and MHC II peptide loading proceeded similarly in all three DC populations. We then analyzed MHC II localization and Ag presentation in CD8+ DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution

Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.

Cystatin C modulates neurodegeneration and neurogenesis following status epilepticus in mouse

Brain damaging insults cause alterations in neuronal networks that trigger epileptogenesis, and eventually lead to the appearance of spontaneous seizures. The present experiments were designed to study the cellular expression and functions of a cysteine proteinase inhibitor, cystatin C, whose gene expression is previously shown to be upregulated in the rat hippocampus during status epilepticus (SE)-induced epileptogenesis. The present data showed that the expression of cystatin C protein increased in the mouse hippocampus 7 days following SE and localized mainly to astrocytes and microglia. Acute neuronal death in the hippocampus at 24 h after SE was reduced in cystatin C-/- mice. Also, the basal level of neurogenesis in the subgranular layer of dentate gyrus was decreased in cystatin C-/- mice compared to wildtype littermates. Interestingly, migration of newly born neurons within the granule cell layer was attenuated in cystatin C-/- mice. These data demonstrate that cystatin C has a role in neuronal death and neurogenesis during SE-induced network reorganization.

CD14(hi) HLA-DRdim macrophages, with a resemblance to classical blood monocytes, dominate inflamed mucosa in Crohn's disease

Intestinal Mϕ play an important role in maintaining gut homeostasis. However, little is known about these cells, their precursors, and their role in intestinal inflammation. Here, we characterize the CD14+ mononuclear cell populations in intestinal mucosa and blood in patients with CD. Among the LP CD14+ Mϕ, we identified three distinct HLA‐DR+‐expressing subsets. Compared with uninflamed, inflamed mucosa contained a marked increase in the proportion of the CD14hiHLA‐DRdim cellular subset. This subset resembled the classical blood monocytes with low CD16, HLA‐DR, and CX3CR1 expression. Classical monocytes migrated efficiently toward CCL2 and released the highest levels of MMP‐1 and proinflammatory cytokines when stimulated with immune complexes or LPS. Our findings strongly suggest that it is the classical and not the intermediate or nonclassical monocytes that are the precursors to the dominating intestinal CD14hiHLA‐DRdim subset. This enhances our understanding of CD pathology and may provide new options in treatment.

A major population of mucosal memory CD4(+) T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality.

Mucosal tissues contain large numbers of memory CD4+ T cells that, through T-cell receptor-dependent interactions with antigen-presenting cells, are believed to have a key role in barrier defense and maintenance of tissue integrity. Here we identify a major subset of memory CD4+ T cells at barrier surfaces that coexpress interleukin-18 receptor alpha (IL-18Rα) and death receptor-3 (DR3), and display innate lymphocyte functionality. The cytokines IL-15 or the DR3 ligand tumor necrosis factor (TNF)-like cytokine 1A (TL1a) induced memory IL-18Rα+DR3+CD4+ T cells to produce interferon-γ, TNF-α, IL-6, IL-5, IL-13, granulocyte–macrophage colony-stimulating factor (GM-CSF), and IL-22 in the presence of IL-12/IL-18. TL1a synergized with IL-15 to enhance this response, while suppressing IL-15-induced IL-10 production. TL1a- and IL-15-mediated cytokine induction required the presence of IL-18, whereas induction of IL-5, IL-13, GM-CSF, and IL-22 was IL-12 independent. IL-18Rα+DR3+CD4+ T cells with similar functionality were present in human skin, nasal polyps, and, in particular, the intestine, where in chronic inflammation they localized with IL-18-producing cells in lymphoid aggregates. Collectively, these results suggest that human memory IL-18Rα+DR3+ CD4+ T cells may contribute to antigen-independent innate responses at barrier surfaces.

A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets.

Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies.