Pertussis toxin-induced reversible encephalopathy dependent on monocyte chemoattractant protein-1 overexpression in mice

Abstract

In this report we describe pertussis toxin-induced reversible encephalopathy dependent on monocyte chemoattractant protein-1 (MCP-1) overexpression (PREMO), a novel animal model that exhibits features of human encephalopathic complications of inflammatory disorders such as viral meningoencephalitis and Lyme neuroborreliosis as well as the mild toxic encephalopathy that commonly precedes relapses of multiple sclerosis (MS). Overexpression of the mouse MCP-1 gene product (classically termed JE) in astrocytes, the major physiological CNS cellular source of MCP-1, failed to induce neurological impairment. Unexpectedly, transgenic (tg) mice overexpressing MCP-1 at a high level (MCP-1(hi)) manifested transient, severe encephalopathy with high mortality after injections of pertussis toxin (PTx) plus complete Freund's adjuvant (CFA). Surviving mice showed markedly improved function and did not relapse during a prolonged period of observation. Tg mice that expressed lower levels of MCP-1 were affected minimally after CFA/PTx injections, and tg expression of other chemokines failed to elicit this disorder. The disorder was significantly milder in mice lacking T-cells, which therefore play a deleterious role in this encephalopathic process. Disruption of CC chemokine receptor 2 (CCR2) abolished both CNS inflammation and encephalopathy, identifying CCR2 as a relevant receptor for this disorder. Proinflammatory and type 1 cytokines including TNF-alpha, IL-1beta, IFN-gamma, IL-2, RANTES, and IP-10 were elevated in CNS tissues from mice with PREMO. These studies characterize a novel model of reversible inflammatory encephalopathy that is dependent on both genetic and environmental factors.

Fig. 1.

Establishment of transgenic mice overexpressing MCP-1 directed by GFAP promoter. a, Construct of a human GFAP promoter driving the expression of murine MCP-1. b, Southern blot demonstrating samples of DNA that contain elements of human GFAP promoter. c, Northern blot showing targeted MCP-1 gene expressions specifically in tissue homogenates of brain, spinal cord, and sciatic nerve. Experiments were performed as previously described in Huang et al. (2001). d, Different levels of MCP-1 expressions in tissue homogenates of CNS from three lines of MCP-1 transgenic mice, i.e., MCP-1_hi,_me, and _low. MCP-1 levels were measured according to Karpus et al. (1998). e, Compared with astrocytes from nontransgenic littermate control mice, astrocytes from MCP-1 tg⁺ mice secreted significantly higher levels of MCP-1. Astrocytes were obtained and cultured by following the protocol described previously in Han et al. (2001). Levels of MCP-1 were determined via ELISA kits from R & D Systems. f, Comparable levels of MCP-1 expression in spinal cords from MCP-1_hitg⁺ non-EAE and tg⁻ EAE mice.Lane 1, MCP-1 tg⁻ wild-type mouse with EAE; lanes 2, 3, MCP-1_hitg⁺ with EAE; lanes 4, 6, tg⁻ naive mice; lanes 5, 7, MCP-1_hi tg⁺ with no challenge.

Fig. 2.

PREMO in huGFAP-MCP-1_hitg⁺ mice. Intravenous PTx injections alone or in combination with subcutaneous CFA induced PREMO in huGFAP-MCP-1_hi tg⁺, but not in tg⁻, mice. Shown is time-lapse photography 4 d after PTx/CFA injection revealing hunched posture and lack of exploratory behavior in a tg⁺ mouse compared with the normal tg⁻ mouse.

Fig. 3.

High death rate and clinical course of PREMO in SWXJ huGFAP-MCP-1_hi tg⁺ mice.a, Thirty-two of 56 SWXJ huGFAP-MCP-1_hitg⁺ mice died of PREMO within 2 wk after the onset of PREMO compared with nil in their tg⁻ littermate control mice; p < 0.01. b, PREMO was induced in MCP-1_hi mice, but not in MCP-1_meand _low tg⁺ and tg⁻mice. HuGFAP-MCP-1_hi, _me, and_low tg⁺ and tg⁻ mice were immunized with PTx and CFA, weighed, and scored daily for PREMO. Shown are PREMO scores (mean ± SD) of mice in each group;p < 0.01 compared between MCP-1_hitg⁺ and others (MCP-1_me and_low tg⁺ and tg⁻mice). n = 56, 19, 31, and 45 in groups of MCP-1_hi, _me, and _lowtg⁺ and tg⁻ mice, respectively.

Fig. 4.

CNS inflammation and blood–brain barrier disruption in huGFAP-MCP-1_hi tg⁺ mice with PREMO. Although minute infiltrate was found in CNS from MCP-1_hitg⁺ naive mice (a, b, arrowhead), H&E staining showed large amounts of infiltrates surrounding blood vessels and in the parenchyma in tissues from huGFAP-MCP-1_hitg⁺ mice injected with PTx and CFA in the presence of CCR2 (c, d). Neither significant infiltrate nor perivascular inflammation (arrows indicating vessels) was observed in their huGFAP-MCP-1_hitg⁺ littermate control mice on CCR2 mutant background (e, f). Mice recovered from PREMO contained perivascular infiltrates in the CNS (g, h). Immunoperoxidase histochemistry revealed abundant extravasated IgG in the cerebellar white matter, meninges (g, i), and spinal cord (h, j) of huGFAP-MCP-1_hi tg⁺ mice (g, h), but not tg⁻(i, j) mice, 4 d after receiving injections of PTx plus CFA. Note positive IgG staining in peripheral nerve elements of the tg⁻ control mouse at bottom left. a, c, e, g, Sections of brain stems, original × 100; b, d, f, h, sections of spinal cords, original × 50; i, k, brain sections, original × 25; j, l, spinal cords, original × 100.

Fig. 5.

Flow cytometric analyses of CNS infiltrates in huGFAP-MCP-1_hi tg⁺ mice with PREMO. Shown are high levels of MHC class II antigen expression on numerous leukocytes (CD45^high) isolated from CNS tissues of intracardially perfused MCP-1_hitg⁺·CCR2⁺mice with PREMO (a). Disruption of CCR2 abolishes the influx of leukocytes in MCP-1_hi tg⁺mice with PREMO (b). CNS tissues from MCP-1_hitg⁺·CCR2⁻mice injected with PTx plus CFA contained mainly CD45^low microglia, virtually the same as those from MCP-1 tg⁻ mice injected with PTx plus CFA (data not shown). Albeit with the lower prevalence and milder manifestation (Fig.7, Table 2), PREMO could be induced by injections of PTx plus CFA in MCP-1_hitg⁺·RAG1⁻mice. Note the presence of CD45^high leukocytes and the absence of both CD4⁺ (d) and CD8⁺ (f) T-cells in CNS tissues from MCP-1_hitg⁺·RAG1⁻mice with PREMO compared with those in MCP-1_hitg⁺·RAG1⁺ CNS tissues (c, e). Flow cytometric analyses of CNS cells from CNS tissues of huGFAP-MCP-1_hi tg⁺-untreated mice and their wild-type controls were akin to b.

Fig. 6.

Increased levels of proinflammatory and Th1 cytokines in CNS tissues from huGFAP-MCP-1_hitg⁺ mice with PREMO. a, RPA showing cytokine expression in samples of CNS tissues from MCP-1_hitg⁺ and MCP-1_hi tg⁻mice injected with PTx plus CFA. b, Quantitative analyses of inflammatory cytokine expression. M, Marker;tg, transgenic; +, tg-positive; −, tg-negative.

Fig. 7.

Failure of PREMO induction in huGFAP-MCP-1_hi tg⁺ mice lacking CCR2 and attenuated PREMO in mice deficient for recombination activation gene 1 (RAG1). a, Compared with the high death rate (25 of 46, 54%) in SJL×B6 MCP-1_hitg⁺ mice with intact CCR2, none of the MCP-1_hitg⁺·CCR2⁻and MCP-1_hitg⁺·RAG1⁻mice injected with PTx plus CFA died of PREMO; p < 0.01. b, SJL×B6 MCP-1_hitg⁺ mice developed PREMO similar to that in other strains, e.g., SWXJ (Fig. 4), SWR, and SJL (data not shown). Although MCP-1_hitg⁺·RAG1⁻mice exhibit an attenuated PREMO, MCP-1_hitg⁺·CCR2⁻mice were free from PREMO. Shown are PREMO scores (mean ± SD) in each group. MCP-1_hitg⁺·RAG1⁻mice that remained healthy (18 of 31; see Table 2) were excluded.