How T-cell-dependent and -independent challenges access the brain: vascular and neural responses to bacterial lipopolysaccharide and staphylococcal enterotoxin B

Abstract

Bacterial lipopolysaccharide (LPS) is widely used to study immune influences on the CNS, and cerebrovascular prostaglandin (PG) synthesis is implicated in mediating LPS influences on some acute phase responses. Other bacterial products, such as staphylococcal enterotoxin B (SEB), impact target tissues differently in that their effects are T-lymphocyte-dependent, yet both LPS and SEB recruit a partially overlapping set of subcortical central autonomic cell groups. We sought to compare neurovascular responses to the two pathogens, and the mechanisms by which they may access the brain. Rats received iv injections of LPS (2 microg/kg), SEB (1mg/kg) or vehicle and were sacrificed 0.5-3h later. Both challenges engaged vascular cells as early 0.5h, as evidenced by induced expression of the vascular early response gene (Verge), and the immediate-early gene, NGFI-B. Cyclooxygenase-2 (COX-2) expression was detected in both endothelial and perivascular cells (PVCs) in response to LPS, but only in PVCs of SEB-challenged animals. The non-selective COX inhibitor, indomethacin (1mg/kg, iv), blocked LPS-induced activation in a subset of central autonomic structures, but failed to alter SEB-driven responses. Liposome mediated ablation of PVCs modulated the CNS response to LPS, did not affect the SEB-induced activational profile. By contrast, disruptions of interoceptive signaling by area postrema lesions or vagotomy (complete or hepatic) markedly attenuated SEB-, but not LPS-, stimulated central activational responses. Despite partial overlap in their neuronal and vascular response profiles, LPS and SEB appear to use distinct mechanisms to access the brain.

Figure 1

LPS- and SEB-induced activational responses in vascular cell types. Darkfield photomicrographs of emulsion-coated slides showing vascular expression of mRNAs encoding the immediate-early gene, NGFI-B (top row), and the vascular early response gene, Verge (bottom row), in rats sacrificed 30–60 min after iv injection of vehicle (left), LPS (middle) or SEB (right). From low levels in controls, both pathogens provoke recruitment of vascular cells, with responses to LPS being more robust. Insets in bottom panels show dual localization of the perivascular cell marker, ED2-ir, with NGFI-B and Verge mRNAs. ED-2-ir perivascular cells (brown) and NGFI-B (top) or Verge (bottom) mRNAs (black grains) around blood vessels. Some NGFI-B mRNA-positive cells manifest ED2-ir (black arrows), but none of the ED2-ir cells (red arrowheads) display Verge mRNA signal (blue arrowheads), indicating a non-macrophage phenotype of Verge-positive cells. Scale bars: 100 μm.

Figure 2

LPS- and SEB-induced COX-2 mRNA and protein in vascular cells. Upper panels: Darkfield micrographs of emulsion coated slides showing COX-2 mRNA induced in CNS blood vessels from rats sacrificed 1hr after iv injection of LPS (2 μg/kg; left) or SEB (1 mg/kg; right). Bottom: Brightfield micrographs showing vascular-associated COX-2-ir from rats sacrificed 3hr after iv LPS (left) or SEB (right) treatment. At doses, LPS (right) elicits enzyme induction in two cell types, a stronger response in multipolar cells (black arrows; previously identified as perivascular cells) and a weaker one in round nuclear cells (red arrows; endothelia). By contrast, SEB (left panel) stimulates COX-2-ir only in multipolar cells (perivascular cells; black arrows). Scale bars: 50 μm.

Figure 3

Effect of graded levels of prostaglandin synthesis blockade on LPS- and SEB-stimulated Fos-ir in the PVH and CeA. Brightfield images of sections through similar levels of the PVH (top) and CeA (bottom) stained for Fos-ir from animals pretreated intravenously with vehicle or 1 or 10 mg/kg indomethacin (Indo) 15 min prior to iv challenge with LPS or SEB. Indomethacin pretreatment produces a dose-related decrease in LPS-induced Fos-ir in PVH, but not CeA. By contrast, nonselective COX inhibition affects neither the low level of SEB-induced Fos expression in PVH nor the more robust response in the amygdala. Other abbreviations: CeAm, l, medial, lateral parts of central nucleus of the amygdala; dp, dorsal parvocellular part of PVH; mpd, v, dorsal, ventral medial parvocellular parts of PVH; pm, posterior magnocellular part of PVH. Scale bars: 100μm.

Figure 4

SEB- and LPS-induced activational responses in select CNS regions of normal animals (NB) and of animals treated with 10 mg/kg indomethacin (Indo). Mean ± SEM number of Fos-ir neurons in the indicated cell group of rats killed 3 hours after iv injection of saline (open bars), 1 mg/kg SEB, or 2 μg/kg LPS in normal animals (black bars) or animals pre-treated with vehicle (V) or 10 mg/kg indomethacin (gray bars). Pre-treatment with indomethacin markedly reduced LPS-induced Fos-ir in a subset of central autonomic cell groups (PVH, NTS and VLM). By contrast, no reliable effect of cyclooxygenase blockade was detected in any of the regions examined in SEB-treated animals. *, differs significantly from saline-injected controls at P<0.05; **, P<0.01; ***, p<0.001. ^†, differs reliably from respective vehicle-pretreated controls at P<0.05; ^††, P<0.01; ^†††, p<0.001. ⁿ, non-significant (P>0.05).

Figure 5

Liposome treatment effects on PVH and CeA responses to LPS and SEB. Fos-ir in the PVH and CeA of rats treated with clodronate liposomes and subsequently challenged with intravenous LPS (top and middle panels) or SEB (bottom panel) and killed 3hr later. Relative to control levels (see Fig. 4) clodronate-liposome treatment enhances PVH responses to LPS (note higher density of activated neurons in the magnocellular region; pm), while not affecting that in the CeA. By contrast, liposome-mediated macrophage ablation does not modify SEB-induced responses in CeA or in PVH (not shown). Other abbreviations as in Fig. 3. Scale bars: 100 μm.

Figure 6

Confirmation of area postrema lesions and abdominal vagotomy. Top: Brightfield photomicrographs of Nissl-stained sections through the level of area postrema, showing the appearance of the region in a sham-operated control (Intact) and in a rat that underwent aspiration of the area postrema (AP). Bottom: Brightfield photomicrographs showing the robust retrograde labeling (immunopositive signal for True Blue) displayed by vagally intact rats of cells in the dorsal motor nucleus of the vagus (DMX; arrows) resulting from retrograde tracer injections placed in the wall of the stomach at the time of surgery, and the complete lack of such labeling in a rat subjected to complete abdominal vagotomy. Other abbreviations: cc, central canal; NTS, nucleus of the solitary tract; XII, hypoglossal nucleus. Scale bars: 200μm.

Figure 7

Area postrema lesions modify Fos expression in SEB-, but not LPS-responsive central autonomic cell groups. Brightfield photomicrographs showing sections through comparable levels of the PVH (top), CeA (middle) and PBl (bottom) stained for Fos-ir from sham-operated controls (Sham) and animals that sustained aspiration lesions of the area postrema (APX) were subsequently challenged with 1mg/kg SEB (left grouping) or 2 μg/kg LPS (right). Ablation of the area postrema markedly attenuated SEB-induced Fos-ir in the CeA and PBl, but reduced LPS-induced activational responses only in the PVH. Other abbreviations: CeAl, m, lateral, medial parts of the central nucleus of the amygdala; cl, central lateral parabrachial nucleus; el, external lateral parabrachial nucleus. Scale bars: 100μm.

Figure 8

Lesion effects on SEB- and LPS-induced activation of representative central autonomic cell groups. Mean ± SEM number of Fos-ir neurons in the CeA and PVH of rats killed 3 hours after iv injection of saline (open bar), 1 mg/kg SEB or 2 μg/kg LPS in sham operated rats (C; black bars) or animals that underwent area postrema lesions (APX) or complete (VX) or selective hepatic (hVX) vagotomy (gray bars). Each of these manipulations significantly reduced SEB-induced Fos expression in the CeA. The only reliable effect on LPS effects was an attenuation of PVH activation in area postrema lesioned rats. *, differs significantly from saline-injected controls at P<0.05; **, P<0.01; ***, p<0.001. ^†, differs reliably from respective surgical controls at P<0.05; ^††, P<0.01; ^†††, p<0.001. ⁿ, non-significant (P>0.05).

Figure 9

Vagotomy modifies SEB-, but not LPS-, induced Fos expression in central autonomic cell groups. Top two rows: Brightfield photomicrographs showing sections though comparable levels of the PVH and CeA stained for Fos-ir from animals that underwent sham operations (Sham) or complete subdiaphragmatic vagotomy (VX) (lower two rows) three weeks prior to intravenous challenge with 2 μg/kg LPS (left) or 1mg/kg SEB (right). Bottom two rows: Arranged similarly, but showing sections through the CeA and BSTov from rats that received sham or selective hepatic vagotomies (hVX) prior to LPS or SEB challenge. Either complete subdiaphragmatic vagotomy or selective hepatic branch vagotomy markedly attenuated SEB-induced Fos-ir in the CeA and BSTov, but did not affect LPS-induced activational responses in either the PVH, CeA or BSTov. Other abbreviations: ac, anterior commissure; ic, internal capsule; lv lateral ventricle. Scale bars: 100μm.

Figure 10

Schematic summary on a mid-sagittal image of rat brain to illustrate circuits and mechanisms implicated as underlying central autonomic activational responses elicited by systemic LPS (red) versus SEB (blue) challenges. The present and previous data support a role for cerebrovascular transduction of LPS signals, with resultant prostanoid synthesis driving engagement of the NTS and VLM, and, consequently, targets of their axonal projections in the hypothalamus (PVH). By contrast, a circumventricular organ (CVO; the area postrema (AP)), and/or peripheral neural (vagal) mechanisms are implicated in driving responses of a distinct subset of central autonomic cell groups (BSTov, CeA, PBl) to SEB. Although these same structures are also responsive to LPS, none of the manipulations attempted here affected their recruitment by LPS, leaving its basis an open question.