Innate Immunity

Erythrocytes have been suggested to bind chemokines, thus limiting their effective concentration in the circulation. We investigated binding of the chemokines IL-8 and RANTES to erythrocytes following endotoxin challenge. Endotoxin caused a transient increase of IL-8 in plasma, and a gradual increase of erythrocyte-bound IL-8, remaining elevated for at least 24 h in vivo. Binding of IL-8 to erythrocytes was confirmed by incubation of freshly isolated erythrocytes with endotoxin stimulated plasma. Incubation for 1 h led to a decrease in plasma concentrations of IL-8 with a concomitant increase in erythrocyte-bound IL-8 levels. Erythrocyte-bound RANTES was induced early following endotoxin infusion and our data may suggest competition between the two chemokines for the same binding site on erythrocytes in vivo. In conclusion, in humans, both IL-8 and RANTES are induced by endotoxin and are, at least in part, cleared from the plasma by binding to erythrocytes.

The presence of functional lipopolysaccharide binding protein (LBP) was investigated in cerebrospinal fluid (CSF) samples of patients with neurological disorders. LBP was detected in CSF by RIA and by a bioassay that measures the ability of LBP to present LPS to human monocytes. Mean concentration of LBP was 1.72 μg/ml in normal CSF, i.e. 1/10 of the concentration measured in normal plasma. LBP concentration was found to be increased in CSF of patients with neurological disorders associated with increased protein levels in CSF, in patients with meningitis/encephalitis and with compressing tumors. LBP levels correlated with protein levels, but not with the clinical presentation. Even in normal CSF, LBP was present in concentrations sufficient to trigger maximal activation of monocytes upon LPS challenge, as measured by tumor necrosis factor induction of mRNA and protein synthesis. The role played by LBP in CSF remains to be determined. However, because it is present and functionally fully active, LBP could play a detrimental role in CSF in the presence of low concentrations of LPS during episodes of Gram-negative infections.

The smooth (S)-form lipopolysaccharides (LPSs) having long O-polysaccharides were successfully isolated by centrifugal partition chromatography from the co-existing rough (R)-form species from the LPSs of wild-type strains of Salmonella enterica, serovars abortus equi and friedenau, and from Citrobacter freundii. Separation is based on different partition behaviors of the R- and S- form LPS species between aqueous and organic phases. The procedure described proved to be convenient, reproducible and readily applicable to large amounts of various LPS. It is another tool to study the relationship between chemical structure and biological activity of LPS.

Myeloid-derived suppressor cells (MDSCs) are notable innate immune cells, which are further divided into two subpopulations, i.e., monocytic and granulocytic. These cells are traditionally considered to mainly suppress the T-cell responses. However, more updated data indicate that their properties are rather immunomodulatory than solely immunosuppressive. Indeed, MDSCs display extensive crosstalk with other either innate or adaptive immune cells, and, according to the situation under which they are triggered, they may enhance or attenuate the immune response. However, their positive role in host's defense mechanisms under specific conditions is rarely discussed in the literature. In this mini-review, the authors briefly summarise the mechanisms of action of MDSCs under distinct conditions, such as infections and malignancies, with a particular emphasis on their role as components of the innate immunity system.

Fibromyalgia (FM) is a syndrome characterized by widespread chronic pain and is associated with elevated systemic inflammatory biomarkers, and an elevated innate cellular response. The aim of this study was to determine if fibromyalgia patients have altered ability to release pro-inflammatory chemokines by isolated neutrophils and monocytes. The study participants were women diagnosed with FM (n = 6) and a control group of healthy women (HW) (n = 6). Supernatant concentrations of eotaxin (CCL11), human macrophage-derived chemokine (MDC) (CCL22) and growth regulated-oncogene (GRO-α) (CXCL1) released by both monocytes and neutrophils either resting or stimulated by LPS were determined by ELISA and compared between the FM and HW groups. Both resting and activated monocytes from FM patients released more eotaxin, MDC and GRO-α than those from HW. However, there were no significant differences in the release of chemokines from neutrophils of FM patients and the ones from healthy women. In conclusion, monocytes from women with FM are deregulated, releasing higher amounts of eotaxin, MDC and GRO-α than healthy individuals. This fact does not occur in neutrophils from women with FM.

Muramyldipeptide (MDP), the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2). Muramyldipeptide enhances the activities of lipopolysaccharide (LPS), but the mechanism underlying this effect is unclear. Here, we obtained evidence that intravenously injected MDP augments LPS-induced hypothermia in wild-type mice, but not in mice deficient in interleukin (IL)-1α/β and/or tumor-necrosis factor (TNF)-α. Muramyldipeptide also: (i) increased pro-IL-1β in tissues, but did not increase IL-1β in serum (since caspase-1 was not activated by MDP); (ii) downregulated the expression of suppressor of cytokine signaling 1 (SOCS1; a negative-feedback regulator of LPS-induced signaling); and (iii) augmented the LPS-induced production of TNF-α, IL-12 p40, and interferon (IFN)-γ. Moreover, by performing in vivo and in vitro experiments, we obtained evidence that macrophages were involved in these effects of MDP. These results suggest that two different mechanisms may underlie the augmenting effect of MDP: namely, stimulation of pro-IL-1β production by, and down-regulation of SOCS1 in, macrophages. We consider that this work may help to elucidate the pathogenesis of mixed bacterial infections, including septic shock and multiple organ dysfunction syndrome (MODS).

Otitis media (OM), a common infectious disease in children, is associated with bacterial middle ear (ME) infection. Toll-like receptors (TLRs) are important mediators of innate immune responses, and TLR9 specifically recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motifs in bacterial DNA. Additional sensors of foreign DNA have recently been identified. The role of DNA sensing and TLR9 was investigated in a murine model of OM induced by non-typeable Haemophilus influenzae (NTHi). Expression of genes related to DNA-sensing pathways involved in innate immunity was assessed via DNA microarray, qPCR and immunohistochemistry. Middle ear responses to NTHi were examined in wild-type and TLR9^−/− mice by histopathology and bacterial culture. Expression of TLR9 signaling genes was modestly up-regulated during OM, as was TLR9 protein in both ME mucosal cells and infiltrating leukocytes. However, genes known to be regulated by CpG DNA were dramatically up-regulated, as were genes involved in DNA sensing by DIA, Pol-III and AIM2. Toll-like receptor 9 deletion significantly prolonged the inflammatory response induced by NTHi in the ME and delayed bacterial clearance. The results suggest that DNA sensing via TLR9 plays a role in OM pathogenesis and recovery. Alternative forms of DNA sensing may also contribute to OM.

A blood luminescence system (BLS) was employed to analyze blood phagocyte function in response to infusion of endotoxin (4 ng Escherichia coli lipopolysaccharide (LPS)/kg body weight) into 7 healthy human subjects. The subjects were closely monitored clinically, and extensive chemical, hematological and coagulation measurements were taken during the pretreatment, early (symptomatic, 1—8 h post-LPS), and late (asymptomatic, 12—48 h post-LPS) phases of acute inflammation. BLS assessment included measurement of basal and PMA-stimulated phagocyte oxidase and myeloperoxidase (MPO) activities, and also included measurement of circulating (COR) and PAF-primed maximum (MOR) opsonin receptor-dependent phagocytic activities. Basal oxidase activity peaked at T+1 h and showed an additional peak at T+24 h post-LPS. The COR activity also peaked at 1—2 h, but remained elevated through T+24 h post-LPS, while the basal MPO activity peaked only once at T+1 h. We concluded that while MPO evidence of phagocyte respiratory activation returned to baseline by T+ 4 h, COR evidence of receptor expression (receptor alert) remained elevated through T+24 h. During this early (0—8 h) period, elastase/α 1AT complex concentration peaked at T+3—4 h and again at T+8 h. Peak numbers of circulating polarized and vacuolated phagocytes also appeared at T+3 h and 7 h. We concluded that there was biochemical and morphological evidence of continuing phagocyte activity beyond T+4 h to T+8 h, and that this corresponded with the period during which the subjects were symptomatic. In addition, the appearance of a second peak of basal oxidase activity at T+24 h, multiple discriminant analyses of all the luminescence data, and the sustained elevation of lactate suggested that there was a later second stage (T+12 h to 48 h) of the human response to endotoxin, during which time the subjects were asymptomatic.

A detailed knowledge of LPS biosynthesis is of the utmost importance in understanding the function of the outer membrane of Gram-negative bacteria. The regulation of LPS biosynthesis affects many more compartments of the bacterial cell than the outer membrane and thus contributes to the understanding of the physiology of Gram-negative bacteria in general, on the basis of which only mechanisms of virulence and antibiotic resistance can be studied to find new targets for antibacterial treatment. The study of LPS biosynthesis is also an excellent example to demonstrate the limitations of `genomics' and `proteomics', since secondary gene products can be studied only by the combined tools of molecular genetics, enzymology and analytical structural biochemistry. Thus, the door to the field of `glycomics' is opened.

Endotoxin is considered to be a systemic (immunological) stressor eliciting a prolonged activation of the hypothalamo-pituitary-adrenal (HPA) axis. The HPA-axis response after an endotoxin challenge is mainly due to released cytokines (IL-1, IL-6 and TNF-α) from stimulated peripheral immune cells, which in turn stimulate different levels of the HPA axis. Controversy exists regarding the main locus of action of endotoxin on glucocorticoid secretion, since the effect of endotoxin on this neuro-endocrine axis has been observed in intact animals and after ablation of the hypothalamus; however, a lack of LPS effect has been described at both pituitary and adrenocortical levels. The resulting increase in adrenal glucocorticoids has well-documented inhibitory effects on the inflammatory process and on inflammatory cytokine release. Therefore, immune activation of the adrenal gland by endotoxin is thought to occur by cytokine stimulation of corticosteroid-releasing hormone (CRH) production in the median eminence of the hypothalamus, which, in turn stimulates the secretion of ACTH from the pituitary. Acute administration of endotoxin stimulates ACTH and cortisol secretion and the release of CRH and vasopressin (AVP) in the hypophysial portal blood. During repeated endotoxemia, tolerance of both immune and HPA function develops, with a crucial role for glucocorticoids in the modulation of the HPA axis. A single exposure to a high dose of LPS can induce a long-lasting state of tolerance to a second exposure of LPS, affecting the response of plasma TNF-α and HPA hormones. Although there are gender differences in the HPA response to endotoxin and IL-1, these responses are enhanced by castration and attenuated by androgen and estrogen replacement. Estrogens attenuate the endotoxin-induced stimulation of IL-6, TNF-α and IL-1ra release and subsequent activation in postmenopausal women. There appears to be a temporal and functional relation between the HPA-axis response to endotoxin and nitric oxide formation in the neuro-endocrine hypothalamus, suggesting a stimulatory role for nitric oxide in modulating the HPA response to immune challenges.

This study investigated the influence of the cholinergic system on neuro-inflammation using nicotinic and muscarinic receptor agonists and antagonists. Intracerebroventricular (ICV) injection of lipopolysaccharide (LPS, 50 µg) was used to induce neuro-inflammation in rats and estimations of pro-inflammatory cytokines, α7 nicotinic acetylcholine receptor (nAChR) mRNA expression were done in striatum, cerebral cortex, hippocampus and hypothalamus at 24 h after LPS injection. Nicotine (0.2, 0.4 and 0.8 mg/kg, i.p.) or oxotremorine (0.2, 0.4 and 0.8 mg/kg, i.p.) were administered 2 h prior to sacrifice. We found that only nicotine was able to block the proinflammatory cytokines induced by LPS whereas, oxotremorine was found ineffective. Methyllycaconitine (MLA; 1.25, 2.5 and 5 mg/kg, i.p.), an α7 nAChR antagonist or dihydro-β-erythroidine (DHβE; 1.25, 2.5 and 5 mg/kg, i.p.), an α4β2 nAChR antagonist, was given 20 min prior to nicotine in LPS-treated rats. Methyllycaconitine antagonized the anti-inflammatory effect of nicotine whereas DHβE showed no effect demonstrating that α7 nAChR is responsible for attenuation of LPS-induced pro-inflammatory cytokines. This study suggests that the inhibitory role of the central cholinergic system on neuro-inflammation is mediated via α7 nicotinic acetylcholine receptor and muscarinic receptors are not involved.

The hemodynamics of septic shock after endotoxinemiais influenced by the plasma kallikrein/kinin and the renin angiotensin systems. In recent years, new information has improved understanding of the protein/biologically active peptide interactions between these two systems. The plasma kallikrein/kinin system, more commonly known as the contact system, has undergone a re-evaluation as to how it assembles on cell membranes for physiological and pathophysiological activation and as to its role in Gram-negative sepsis. It has been proposed that it counterbalances the plasma renin angiotensin system. Furthermore, more knowledge about the renin angiotensin system has become available on how it either opposes the actions of the kallikrein/kinin system or, in some cases, summates with it. Understanding the interactions between these two systems may lead to development of better pharmacological treatments for endotoxin-induced shock.