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Secretariat for Scientific, Legal and Economic Affairs, Department of Clinical Chemistry
Department of Pediatrics, Ullevaal University Hospital
Department of Pediatrics, Rikshospitalet University Hospital, Oslo, Norway
Chemokines are important in regulating leukocyte traffic during infection. We analyzed plasma chemokine levels of monocyte chemoattractant protein (MCP)1, macrophage inflammatory protein (MIP)1, interleukin (IL)8, and RANTES in patients with meningococcal infection and correlated these to plasma lipopolysaccharide (LPS) levels, which are closely associated with clinical presentation. In patients with fulminant meningococcal septicemia, versus distinct meningitis or mild systemic meningococcal disease, MCP-1 (both P < .0001), MIP-1 (both P < .0001), and IL-8 (P < .0001 and P = .011) were significantly higher and RANTES significantly lower (P = .007 and P = .021). MCP-1 (r = .88), MIP-1 (r = .82), and IL-8 (r = .89) were positively correlated to plasma LPS levels, whereas RANTES was negatively correlated (r = -.49). In an ex vivo whole-blood model, heat-inactivated wild-type Neisseria meningitidis, purified meningococcal LPS, and (to a negligible extent) heat-inactivated LPS-deficient mutant N. meningitidis induced these chemokines. N. meningitidis LPS is the major cause of chemokine release in meningococcal disease.
Chemoattractant cytokines, or chemokines, play an important role in the recruitment and regulation of the leukocyte traffic during acute inflammatory responses [1]. Chemokines are structurally homologous proteins with molecular masses between 6 and 14 kDa, and they are divided into 4 subfamilies (CC, CXC, CX3C, and C) on the basis of the arrangement and number of positionally conserved cysteine motifs. The CC chemokines monocyte chemoattractant protein (MCP)1 (CCL2) and macrophage inflammatory protein (MIP)1 (CCL3) are the major chemoattractants for monocytes during inflammatory responses [2], whereas interleukin (IL)8 (CXCL8), a CXC chemokine, is the major chemoattractant for neutrophil leukocytes [2]. RANTES is a potent chemoattractant for monocytes, eosinophils, and T cells [3].
Chemokine production is induced in monocytes and other hematopoetic cells by different bacterial cell-wall components, such as lipopolysaccharide (LPS) from gram-negative bacteria, lipoteichoic acid from gram-positive bacteria, and lipoarabinomannan from Mycobacterium species, as well as by the inflammatory cytokines tumor necrosis factor (TNF), IL-1, and IL-6 [47].
In meningococcal infections, the levels of key pro- and anti-inflammatory cytokines in plasma are closely associated with the levels of neisserial LPS, clinical presentation, and outcome [812]. IL-8 increases and RANTES decreases in plasma in patients with severe meningococcal septic shock [13, 14]. The cerebrospinal fluid (CSF) of patients with acute bacterial meningitis contains higher levels of MCP-1, MIP-1, and IL-8 than that does of control subjects [15], whereas RANTES was undetectable [16].
Patients with fatal meningococcal septicemia usually have leukopenia at the time of hospital admission [17, 18]. This may reflect an up-regulation of adhesion molecules on leukocytes and endothelial cells by cytokines and chemokines that leads to the margination of circulating leukocytes at the periphery of the blood vessels. Thus, chemokines may play an important role at an early stage in securing the recruitment of white blood cells to inflamed areas in patients with meningococcal septicemia.
The aim of the present study was to examine the plasma levels of 3 different CC chemokines (MCP-1, MIP-1, and RANTES) and the CXC chemokine IL-8 in patients with meningococcal infection and to correlate these chemokine levels to plasma levels of LPS. Furthermore, to elaborate on the importance of Neisseria meningitidis LPS on chemokine induction, we studied and compared the chemokine induction capacities of heat-inactivated wild-type (wt) N. meningitidis with a heat inactivated LPS-deficient mutant of N. meningitidis and with purified LPS from N. meningitidis (Nm LPS) in an ex vivo whole-blood model.
SUBJECTS, MATERIALS, AND METHODS
Subjects.
The study included 69 patients with confirmed meningococcal infection who were divided into 3 groups depending on clinical features: (1) patients with fulminant meningococcal septicemia (n = 24), (2) those with distinct meningitis (n = 28), and (3) those with mild systemic meningococcal disease (n = 17). The plasma samples were analyzed after informed consent was obtained from the patients or their relatives and in accordance with rules approved by the Regional Ethics Committee of Health, region 1, in Norway.
Clinical definitions.
Fulminant meningococcal septicemia was defined as a meningococcal infection that led to rapidly evolving, persistent, severe septic shock with minimal pleocytosis (<100 × 106 leukocytes/L CSF), impaired renal function, and severe coagulopathy. Distinct meningitis was defined as a meningococcal infection with marked CSF pleocytosis (>100 × 106 leukocytes/L CSF) and an absence of septic shock. Mild systemic meningococcal disease was defined as a confirmed meningococcal infection without the development of persistent shock or distinct meningitis. A control group consisting of 10 healthy volunteers was included in the study.
Blood sampling.
Blood from patients was collected at the time of admission into LPS-free heparin tubes (EndoTube ET; Chromogenix) (final heparin concentration in blood, 30 IU/mL). The blood was immediately centrifuged (at 1400 g and 20°C for 10 min), and plasma was pipetted off and stored in aliquots at -70°C. The samples had previously been thawed several times. Blood from the volunteers in the control group was collected under similar conditions.
Purification of Nm LPS.
LPS from reference strain N. meningitidis H44/76 was purified as described by Brandtzaeg et al. [19]. The Nm LPS preparation was reconstituted (1 mg/mL) with LPS-free water, stored at -70°C, and further diluted with LPS-free PBS (pH 7.4) to required concentrations.
Bacterial whole-cell preparations.
N. meningitidis strain H44/76 (LPS+Nm) and the LPS-deficient mutant meningococcal strain H44/76lpxA- (LPS-Nm), which lacks LPS in the outer membrane, were used [20]. The whole-cell preparations were prepared as described by Bjerre et al. [21]. Briefly, the bacteria were grown overnight on GC medium base with isovitalex (Becton Dickinson), harvested into Hanks' balanced salt solution that contained 0.1% (wt/vol) bovine serum albumin, and the number of colony-forming units was determined by measuring the optical density at 630 nm. Heat inactivation of the bacteria was done at 56°C for 30 min. The suspensions contained 1 × 1091 × 1010 cfu/mL, as determined by measuring the optical density at 630 nm, and were further diluted with LPS-free PBS (pH 7.4) to the required number of bacteria. Quantification of the LPS content in the LPS+Nm and LPS-Nm preparations was done by use of SDS-PAGE with silver staining; the purified Nm LPS (H44/76) preparation was used as the standard [22]. No LPS was detected in the LPS-Nm preparations (data not shown).
Ex vivo whole-blood model.
Venous blood (4 mL) was collected from healthy volunteers (n = 3) into sterile polypropylene 4.5-mL tubes (NUNC A/S) that contained heparin (Leo; final concentration in blood, 20 IU/mL). The blood sample was aliquoted (1 mL) into sterile polypropylene tubes (1.8 mL), and a 110-L bacteria suspension (final concentration, 1 × 106 cfu/mL), Nm LPS (final concentration, 0.110 ng/mL), or PBS was added. The blood samples were capped and incubated for 0, 2, 4, or 6 h at 37°C under constant rotation (12 rpm) (MACSmix; Miltenyi Biotec). After incubation, the tubes were centrifuged (at 3000 g and 4°C for 10 min); then, plasma was pipetted off and stored in aliquots at -70°C until analysis within 14 days.
Chemokine quantification.
The levels of MCP-1, MIP-1, IL-8, and RANTES in plasma samples were quantified by ELISA according to the manufacturer's instructions (R&D Systems and Biosource International). Detection limits were 5, 2, 10, and 8 pg/mL, respectively.
Detection of LPS levels.
LPS levels in the patients' plasma samples were quantified by use of the chromogenic Limulus amebocyte lysate (LAL) assay, as described elsewhere [23]. LPS levels were compared with the LAL activity of purified Escherichia coli 055:B5 LPS and expressed as endotoxin units (EU) per milliliter. The detection limit was 0.25 EU/mL.
Statistical analysis.
Patient data are expressed as median values. Statistical significances between the patient groups were analyzed by use of the Mann-Whitney U test (2-tailed). For correlation analysis, the Spearman rank correlation test was used. The data from the ex vivo experiments are given as the mean ± SE. Statistical analysis was performed by use of Student's paired t test. Data were considered statistically significant at a level of P < .05.
RESULTS
Levels of MCP-1, MIP-1, IL-8, and RANTES in patient plasma.
The plasma levels of MCP-1, MIP-1, and IL-8 from patients with fulminant meningococcal septicemia were significantly higher than those in plasma from patients with distinct meningitis (P = .0001, P < .0001, and P < .0001, respectively) or mild systemic meningococcal disease (P < .0001, P < .0001, and P = .011, respectively) and from the control group (P < .0001) (figure 1A1C and table 1). The plasma levels of RANTES from patients with mild systemic meningococcal disease and those with distinct meningitis were significantly higher than those of patients with fulminant meningococcal septicemia (P = .021 and P = .007, respectively) (figure 1D and table 1).
Correlation between plasma chemokine and LPS levels.
Plasma levels of MCP-1, MIP-1, and IL-8 were positively correlated with the plasma levels of LPS (r = .88, P < .0001 [n = 65]; r = .82, P < .0001 [n = 64]; and r = .89, P < .0001 [n = 30], respectively) (figure 2A2C), whereas plasma levels of RANTES showed a negative correlation to plasma levels of LPS (r = -.49, P < .0058 [n = 34]) (figure 2D).
Changes in plasma levels of MCP-1, MIP-1, and IL-8 during antibiotic treatment of fulminant meningococcal septicemia.
Plasma levels of MCP-1 (n = 5), MIP-1 (n = 5), and IL-8 (n = 3) from patients with fulminant meningococcal septicemia were measured during the first 2448 h of treatment. The plasma levels of all 3 chemokines decreased by >50% during the first 26 h of treatment (figure 3A3C).
Ex vivo experiments.
The general pattern of chemokine production (MCP-1, MIP-1, IL-8, and RANTES) in the ex vivo whole-blood model revealed an increase during 6 h of incubation with either LPS+Nm (final concentration, 1 × 106 cfu/mL) or purified Nm LPS (final concentration, 0.1 ng/mL), compared with unstimulated whole blood (figure 4). Chemokine production induced by LPS-Nm (final concentration, 1 × 106 cfu/mL) increased only slightly during 6 h of incubation, compared with the production induced by heparinized whole blood without additional stimuli.
When we examined each chemokine separately, no significant differences were observed between the MCP-1 production induced by LPS+Nm, Nm LPS, and LPS-Nm, but the production induced by LPS+Nm was significantly higher than the production in unstimulated whole blood (P = .04) (figure 4A). MIP-1 production induced by LPS+Nm and Nm LPS was significantly higher than the production induced by LPS-Nm (P = .02) and by unstimulated whole blood (P = .01) (figure 4B). The IL-8 production induced by LPS+Nm was significantly higher than the production induced by purified Nm LPS (P = .02), LPS-Nm (P = .03), and unstimulated whole blood (P = .02) (figure 4C). The production of RANTES was more variable between donors than that of other chemokines. Because of the large variation, RANTES production induced by purified Nm LPS but not by LPS+Nm was significantly higher than the production induced by LPS-Nm (P = .02) and unstimulated whole blood (P = .04) (figure 4D). Pilot experiments that used higher concentrations of Nm LPS (final concentrations, 1, 5, or 10 ng/mL) showed increased production of MIP-1 and especially of IL-8 but no further increase in the production of MCP-1 and RANTES (data not shown).
DISCUSSION
The present study systematically analyzed the plasma chemokine concentrations of MCP-1, MIP-1, IL-8, and RANTES of patients with confirmed meningococcal infection. The results indicate that chemokine levels are correlated to the patients' plasma levels of LPS, which suggests a cause-and-effect relationship. Furthermore, to analyze the importance of Nm LPS in the induction of these chemokines, we have shown the results of an ex vivo whole-blood model, in which these chemokines are induced by LPS+Nm and, for MCP-1, MIP-1, and RANTES, also by Nm LPS. LPS-Nm induced negligible amounts of these chemokines.
The levels of MCP-1, MIP-1, and IL-8 in plasma from patients with fulminant meningococcal septicemia were significantly higher than levels in patients with other clinical presentations. This is in accordance with results from previous studies of both plasma and CSF levels of TNF-, IL-6, and IL-8 in patients with meningococcal disease [8, 9, 13]. In contrast to Sprenger et al. [16], who found elevated chemokine levels in CSF in patients with bacterial meningitis and only occasionally traces of MCP-1 and IL-8 in serum, we consistently detected high levels of MCP-1 and IL-8 in plasma from patients with fulminant meningococcal septicemia. We have previously asserted the view that meningococcal infections are usually compartmentalized, giving rise to increased levels of effector molecules either in the circulation of patients with septicemia or in the CSF of patients with meningitis [24]. The present findings reflect the production in circulation. The presence of chemokines signals for the recruitment of leukocytes to areas of inflammation. Possibly, therefore, the leukopenia regularly observed in meningococcal septicemiaand which is used as a prognostic marker of disease severityshould be viewed as the chemokine-induced margination of white blood cells from the circulation. One may speculate as to the clinical importance of elevated chemokine levels, but studies that have used MIP-1, MCP-1, and CCR2-deficient mice have shown that these chemokines and their receptors play essential roles in mounting adequate responses to inflammation [2527]. The overshoot of the inflammatory responses seen in patients with fulminant meningococcal septicemia may possibly be detrimental to the host [10, 12].
A wide range of different effector molecules have been analyzed in meningococcal disease and have been shown to be highly correlated to plasma LPS levels. These include the cytokines TNF-, IL-1, IL-6, and IL-10 [9, 12, 28]. In addition, we have recently demonstrated, by quantitative measurement of N. meningitidis DNA, that DNA levels in plasma were highly correlated to plasma LPS levels [29]. In concordance with these results, we found that the levels of MCP-1, MIP-1, and IL-8 in patients with meningococcal disease were positively correlated to the plasma LPS levels. Thus, the severity of disease, as evidenced by plasma LPS and meningococcal DNA levels, is reflected in circulating chemokine levels.
The levels of RANTES, however, in plasma presented a different pattern than those of the other chemokines. Patients with mild systemic disease had significantly higher levels of RANTES than patients with fulminant meningococcal septicemia. Furthermore, the levels of RANTES were negatively correlated to the plasma levels of LPS. These results are in accordance with the results published by Carrol et al. [14], who described an inverse relationship among plasma levels of RANTES, severity of disease, and the levels of the proinflammatory cytokines TNF- and IL-8. The role of RANTES in meningococcal disease has not been ascertained, but this chemokine does not seem to play an important role in the recruitment of leukocytes to the subarachnoid space.
The initiation of antibiotic treatment leads to a rapid decrease in plasma levels of LPS, meningococcal DNA, and key inflammatory cytokines [8, 9, 2931]. We observed the same pattern in serial measurements of MCP-1, MIP-1, and IL-8 in the present study. Plasma clearance (a 50% decrease) occurred within 26 h. It thus appears that antibiotic treatment, when it is initiated at an early time point of meningococcal disease, rapidly shuts off microbial growth and aims at restoring homeostasis by clearing effector molecules.
LPS in the outer membrane of N. meningitidis has been considered to be the main inducer of the inflammatory response in patients with meningococcal infection [12, 30, 32]. The construction of LPS-Nm from the serogroup B reference strain H44/76 made it possible for us to study the effects of non-LPS components on cytokine production and other host responses [20]. In the present study, we examined the importance of LPS in the induction of chemokine production. We thus compared the effects of LPS+Nm, LPS-Nm, and Nm LPS. To approach the in vivo patient situation as closely as possible, we used an ex vivo whole-blood model with heparin as an anticoagulant. Pilot experiments showed that heparin did not influence chemokine production, white blood cell count, pH, or the release of the platelet activation marker -thromboglobulin. Furthermore, heparin has consistently been used as an anticoagulant in our patient blood specimens.
LPS+Nm (final concentration, 1 × 106 cfu/mL) induced all 4 chemokines in our model system. LPS-Nm at the same concentration did not induce marked increases in any of the 4 chemokines after 6 h of incubation. However, in previous studies, concentrations of LPS-Nm 10100-fold higher than in the wt strain were required, to obtain the same level of IL-8 and other cytokines [22, 3335]. This indicates that IL-8 can be induced by bacterial cell-wall components other than LPS but that these molecules are weak compared with LPS. By comparing the effect of purified Nm LPS (0.1 ng/mL) with that of LPS+Nm (1 × 106 cfu/mL), we could show that whole bacteria with LPS integrated in the outer membrane were significantly more potent in inducing IL-8, whereas the effects of these 2 inducers were similar to those for the other chemokines investigated. It remains elusive as to why the 2 different preparations have different effects on the production of IL-8, compared with monocytes and lymphocytes, which produce MCP-1, MIP-1, and RANTES. Neutrophils express only 1 of 30 CD14 copies on the surface, compared with monocytes. CD14 is a crucial part of the LPS receptor CD14Toll-like receptor 4MD2 on the surface of LPS-responsive cells [36]. The difference in density of CD14 may contribute to the observed difference in response. The physiochemical presentation of aggregated Nm LPS in plasma may also be related to the difference in IL-8 production in these experiments.
We conclude that LPS is a major cause of chemokine release in meningococcal disease, and this is reflected in the dose-response patterns observed among the patients. Furthermore, chemokine levels of MCP-1, MIP-1, and IL-8 in plasma are directly related to bacterial load, as evidenced by LPS levels (LAL assay) in patient plasma samples. In contrast, however, the levels of RANTES in plasma were inversely related to the levels of LPS. Finally, in an ex vivo whole-blood model that used LPS+Nm, LPS-Nm, or Nm LPS, we have shown that LPS is essential for chemokine release.
Acknowledgments
We thank Petter van der Ley (Rijksinstituut voor Volksgezondheit en Milieu, Bilthoven, The Netherlands), for providing the Neisseria meningitidis H44/76lpxA- knockout mutant; Arne Hiby (Department of Bacteriology, National Institute of Public Health, Oslo), for the whole-cell preparation of N. meningitidis; and Reidun vsteb (The Research and Development Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo), for the detection of lipopolysaccharide in the patients' plasma samples.
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