YKL-40, a marker of simian immunodeficiency virus encephalitis, modulates the biological activity of basic fibroblast growth factor

Abstract

Human immunodeficiency virus encephalitis causes dementia in acquired immune deficiency syndrome patients. Using proteomic analysis of postmortem cerebrospinal fluid (CSF) and brain tissue from the simian immunodeficiency virus primate model, we demonstrate here a specific increase in YKL-40 that was tightly associated with lentiviral encephalitis. Longitudinal analysis of CSF from simian immunodeficiency virus-infected pigtailed macaques showed an increase in YKL-40 concentration 2 to 8 weeks before death from encephalitis. This increase in YKL-40 correlated with an increase in CSF viral load; it may therefore represent a biomarker for the development of encephalitis. Analysis of banked human CSF from human immunodeficiency virus-infected patients also demonstrated a correlation between YKL-40 concentration and CSF viral load. In vitro studies demonstrated increased YKL-40 expression and secretion by macrophages and microglia but not by neurons or astrocytes. We found that YKL40 displaced extracellular matrix-bound basic fibroblast growth factor (bFGF) as well as inhibited the mitogenic activity of both fibroblast growth factor receptor 1-expressing BaF3 cells and bFGF-induced axonal branching in hippocampal cultures. Taken together, these findings demonstrate that during lentiviral encephalitis, YKL-40 may interfere with the biological activity of bFGF and potentially of other heparin-binding growth factors and chemokines that can affect neuronal function or survival.

Figure 1

Comparison of mass spectra from CSF between SIV-infected macaques with and without encephalitis using Q10 ProteinChips shows differences in signal intensities of protein peaks. Representative spectral peaks of SIV-infected macaque with encephalitis (SIVE) (top) and SIV-infected macaque without encephalitis (SIV) (bottom). The x axis shows the mass-to-charge (m/z) ratio in Da (36,000 to 40,000 Da), and the y axis shows the relative abundance (peak intensities). A mass peak of 38,200 Da was evident at significantly increased levels in SIVE macaques.

Figure 2

Longitudinal YKL-40 levels in CSF and plasma from SIV-infected pigtailed macaques. A: Western blot analysis of YKL-40 and β-actin loading control in midfrontal cortical gray matter from noninfected macaques (lanes 1 to 4) and macaques without (lanes 5 to 8) and with (lanes 9 to 12) SIVE. B: CSF from noninfected, SIV-infected, and SIV with encephalitis were analyzed for YKL-40 using the Metra YKL-40 ELISA kit. CSF YKL-40 was significantly higher in SIVE than SIV (P = 0.0002). Longitudinal CSF (C) and plasma (D) samples were drawn every 2 weeks and analyzed for YKL-40 ELISA. E: CSF from HIV-infected patients categorized on the basis of CSF HIV RNA copies was analyzed for YKL-40 by ELISA. CSF YKL-40 was significantly higher in patients with viral load higher than 10,000 copies/ml than patients with lower copies/ml (P = 0.0003). Correlation between CSF YKL-40 levels and CSF viral load in SIVE (F) and SIV (G) was quantitated as described in the Materials and Methods. H: CSF YKL-40 correlation with HIVE pathology.

Figure 3

YKL-40 stain co-localizes with macrophage/microglial marker. Mixed glia-neuronal cultures were prepared as described in the Materials and Methods. On day in vitro (DIV) 4 the cultures were double-labeled for YKL-40 (green) and MAP2 (red) or GFAP (red) or CD68 (red).

Figure 4

YKL-40 is secreted from human primary microglia and macrophages but not astrocyte cultures. Human primary microglia, astrocyte, and macrophage cultures were prepared as described in the Materials and Methods. YKL-40 secretion into the culture medium was measured 4 days after plating using the Metra YKL-40 ELISA kit or 2 weeks after excluding M-CSF from the media for macrophages. One μg/ml of LPS and 1 mmol/L dibutyryl cyclic adenosine monophosphate (DBcAMP) were added to microglia or astrocyte cultures, respectively, 48 hours before assay performance.

Figure 5

YKL-40 macrophage staining in human primary cultures and microglia. Human primary microglia and macrophage cultures were prepared as described in the Materials and Methods. On DIV 4 the cultures were double-labeled with YKL-40 (green) and CD68 (red). Scale bar = 50 μm.

Figure 6

YKL-40 localizes around astrocytes in regions of microglial nodules in SIVE. Double-label immunofluorescence for YKL-40 (green) and CD68 (red) or GFAP (red). Co-localization of YKL around GFAP-positive astrocytes is evident in the yellow signal around cell bodies.

Figure 7

YKL-40 localizes around astrocytes in regions of microglial nodules and occasional CNS activated macrophages/microglia in HIVE. Triple-label immunofluorescence for YKL-40 (green) and CD68 (blue) or GFAP (red). Co-localization of YKL-40 and astrocytes appears as yellow halo around cell soma. Co-localization of YKL with CD68-positive macrophages is evident by aqua signal (arrowheads). Scale bar = 50 μm.

Figure 8

YKL-40 is distributed around activated astrocyte cell bodies in regions with microglial nodules. Six different astrocytes double-label for YKL-40 (green) and GFAP (red). See Supplemental Movie at http://ajp.amjpathol.org for the spatial deposition of YKL-40 around the astrocyte soma.

Figure 9

YKL-40 mRNA co-localizes with microglia/macrophages. Six-μm-thick paraffin-embedded section were stained with CD68 (A) and then hybridized with ³⁵S-labeled RNA probes for YKL-40 (B) as described in the Materials and Methods. C: Grains predominantly localize with CD68-positive cell bodies.

Figure 10

YKL-40 displacement of ECM-bound bFGF. ECM-coated wells were incubated with ¹²⁵I-bFGF. The ECM was then washed to remove the unbound bFGF, and incubated with increasing concentrations of heparin, bovine serum albumin, or YKL-40. Released ¹²⁵I-bFGF was counted in a γ-counter. Released radioactivity is expressed as percentage of total ECM-bound ¹²⁵I-bFGF. Each data point is the mean of duplicate wells (±SEM). The results are representative of at least three different experiments.

Figure 11

YKL-40 inhibits the mitogenic activity of bFGF in FGFR1-expressing BaF3 cells. Proliferation of FGFR1-expressing cells was evaluated using Promega’s CellTiter kit. The cells were incubated with 5 ng/ml of bFGF and the indicated concentrations of heparan sulfate and purified YKL-40. Heparan sulfate and YKL-40 were mixed and incubated on ice for 1 hour before their addition. Each point is the mean value of triplicate samples (±SEM). The results are representative of at least three different experiments.

Figure 12

YKL-40 inhibits bFGF-induced axonal branching of hippocampal neurons. Hippocampal cultures were prepared from E18 Sprague-Dawley pregnant rats and plated on coverslips on astrocyte feeder layer for 2 days. After 2 days the coverslips were transferred to a new plate and grown in serum-free N2 medium without the feeder layer for an additional 2 days in the absence or presence of bFGF (10 ng/ml), heparin (1 ng/ml), and YKL (12.5 μg/ml) as indicated. On day 4 in culture the cells were fixed and labeled with tau. Axonal quantification was done as described in the Materials and Methods. The number of axonal branch points per neuron was counted. A one-way analysis of variance with posthoc comparison via Tukey’s honestly significant difference was used to evaluate between group differences.