* Department of Internal Medicine, University Medical Center Nijmegen, Nijmegen, and
National Institute of Public Health and the Environment, Bilthoven, The Netherlands; and
Institute of Child Health, University College London Medical School, London, United Kingdom
Correspondence: Marcel van Deuren, Department of Internal Medicine, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: m.vandeuren{at}AIG.AZN.NL
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and interleukin (IL)-1ß production and virtually all interferon
(IFN)-
production. Based on tricine sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis of LPS in
proteinase K-treated lysates of N. meningitidis H44/76, a
quantitative comparison was made between the cytokine-inducing capacity
of isolated and purified LPS and LPS-containing meningococci. At
concentrations of >107 bacteria/mL, intact bacteria were
more potent cytokine inductors than equivalent amounts of isolated LPS,
and cytokine induction by non-LPS components was additive to that by
LPS. Experiments with mice showed that non-LPS components of
meningococci were able to induce cytokine production and mortality. The
principal conclusion is that non-LPS parts of N.
meningitidis may play a role in the pathogenesis of meningococcal
sepsis by inducing substantial TNF-, IL-1ß, and IFN-
production.
Key Words: LPS-deficient meningococci • meningococcal sepsis • outer membrane • 2-keto-3-deoxyoctanate • TSDS-PAGE
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-LPS strategies explored so far have failed to ameliorate the clinical course of Gram-negative sepsis [3 4 5 6 ], which raises the question whether LPS is the sole toxic element in Gram-negative sepsis [7 ].
Fulminant meningococcal sepsis is considered the prototypical human gram-negative septic shock, characterized by extremely high endotoxin and cytokine concentrations [8 9 10 11 ]. Thus, the causative bacterium Neisseria meningitidis is a suitable subject for the study of cytokine induction by gram-negative bacteria. Recently, a viable N. meningitidis mutant was constructed that is devoid of LPS but still contains all other outer membrane constituents [12 ]. This mutant has made it possible to assess the cytokine-inducing potency of the non-LPS parts of this gram-negative bacterium in a quantitative fashion.
The principal aim of the present study is to determine the contribution of LPS and non-LPS components of N. meningitidis to the pathogenesis of meningococcal sepsis. Therefore, we compared quantitatively the cytokine production induced by meningococcal LPS, wild-type meningococci, and LPS-deficient meningococci.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Outer membrane complexes (OMCs) of both meningococcal strains were prepared by sarcosyl extraction as described previously [18 ]. These OMCs consist primarily of PorA (class 1), PorB (class 3), and the RmpM (class 4) outer membrane proteins. The pLAK33 OMCs contain no LPS and express increased amounts of an OpA protein [19 ], H44/76 OMCs contain 10–20% LPS. Protein content was determined by a bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard.
The molecular mass of meningococcal strain H44/76 LPS is 4,044 Da [20 ]; one molecule of LPS contains two molecules of KDO (molecular mass, 238 Da). LPS was isolated by the phenol/water extraction method as described by Westphal and Jann [21 ]. After isolation, LPS was treated with proteinase K (Sigma-Aldrich Co.) and with DNase and RNase (Roche Diagnostics) for additional purification, recovered by ultracentrifugation, freeze-dried, and solved in sterile PBS. The amount of protein contamination in this purified LPS solution was determined by bicinchoninic acid assay. DNA and RNA contamination was determined by spectrophotometry using a Genequant RNA/DNA calculator (Pharmacia Biotech AB, Uppsala, Sweden).
The KDO content of the purified H44/76 LPS solution and of the bacterial suspensions was measured spectrophotometrically by the method described by Weissbach and Hurwitz [22 ].
TSDS-PAGE followed by silver staining of LPS was used for quantification of LPS in N. meningitidis H44/76. Cell lysates of H44/76 meningococci were made by suspending 1.35 x 1010 bacteria in 500 µL of SDS-buffer (7.5% glycerol, 1.25 M Tris/HCl, 1.5% SDS) and incubating this for 5 min at 100°C. Proteins in this suspension were digested by incubation with proteinase K (0.5 mg/mL) for 4 h at 56°C [23 ]. Serial dilutions of purified H44/76 LPS were used as a standard. After silver staining, the polyacrylamide gel was analyzed by densitometry.
Blood for the isolation of human peripheral blood mononuclear cells (PBMCs) was drawn in 10-mL EDTA-anticoagulated tubes (Vacutainer System; Beckton Dickinson, Rutherford, NJ) from healthy volunteers. PBMCs were isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia Biotech AB). The cells from the interphase were aspirated, washed three times in sterile PBS, and resuspended in culture medium RPMI 1640 (Dutch modification; Flows Lab, Irvine, Scotland) supplemented with L-glutamine (2 mmol), pyruvate (1 mmol), and gentamycin (50 mg/mL) and 5% freshly pooled human serum. PBMCs (5x106/well) were incubated with various stimuli in 200-µL 96-well plates (Greiner BV, Alphen a/d Rijn, The Netherlands) at 37°C and 5% CO2 for 4 and 24 h. The supernatant was obtained by centrifugation and stored at -20°C until the cytokine assay was performed.
C57Bl/6J mice, obtained from the Jackson Lab (Bar Harbor, ME), were bred in our local facility. The animals were fed standard laboratory food (Hope Farms, Woerden, The Netherlands) and housed under specific pathogen-free conditions; 6- to 8-week-old mice weighing 20 to 25 g were used for the experiments. Resident peritoneal macrophages were harvested by rinsing the peritoneal cavity aseptically with sterile PBS (4°C), 105 cells/well were incubated with the different stimuli for 24 h. For lethality experiments, mice were pretreated with galactosamine (10 mg/mouse) 30 min before the pathogen was injected into an orbital vein.
Tumour necrosis factor (TNF-) and interleukin-1ß (IL-1ß)
were determined by radioimmunoassay as described previously
[24
]. Interferon- (IFN-) was determined by
enzyme-linked immunosorbent assay with a commercially available kit
(Pelikine Compact Human IFN- ELISA kit, Central Laboratory of the
Netherlands Red Cross, Amsterdam, The Netherlands). The lower detection
limit was 80 pg/mL for both TNF- and IL-1ß and 4 pg/mL for
IFN-. Murine TNF- (mTNF-) and murine IL-1ß (mIL-1ß) were
determined by radioimmunoassay as described by Netea et al.
[25
]. Detection limits were 40 pg/mL for mTNF- and 20
pg/mL for mIL-1ß.
Results were compared by a Wilcoxson-Mann-Whitney test for unpaired, nonparametrical data. Spearman’s rank correlation coefficient was calculated to quantify the correlation between study parameters. P values of >0.05 were considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein contamination in the purified H44/76 LPS was <2.5%, and DNA
and RNA contamination was 3.0% and 2.7%, respectively. This indicates
that the H44/76 LPS used was at least 92% pure. The amount of KDO that
was detected in 1 mg/mL of H44/76 LPS solution was 0.091 mg/mL.
Assuming an average molecular mass for LPS of 4,044 Da, the amount of
LPS in this solution based on the KDO assay was
(0.091x4,044)/(238x2) 
0.8 mg of LPS/mL.
TSDS-PAGE with silver stain of LPS was used to determine the amount of LPS in cell lysates of N. meningitidis H44/76 (Fig. 1 ) [16 , 23 , 26 ]. Density analysis of the silver stain of LPS in different dilutions of cell lysates of N. meningitidis H44/76 bacteria and of serial dilutions of purified LPS showed that 7 x 105 bacteria contain approximately 1 ng of LPS.
 View larger version (31K): [in a new window] |
Figure 1. TSDS-PAGE silver staining of 0.1 µg (lane 1), 0.19 µg (lane
2), 0.39 µg (lane 3), and 0.77 µg (lane 4) of purified H44/76 LPS
and LPS in proteinase K-treated lysates of 5x108 (lane 5)
and 2.5 x 108 (lane 6) N. meningitidis
H44/76 bacteria. Insert: Standard curve as calculated by linear
regression for contour density (OD xmm2)
of the purified LPS samples. Contour density of the lysates
corresponds with 0.7 µg of LPS in sample 5 and 0.34 µg of LPS in
sample 6.
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Quantitative comparison of cytokine induction
The dose-response relationship of meningococcal LPS, wild-type
N. meningitidis H44/76, and LPS-deficient N.
meningitidis pLAK33 for cytokine production by PBMCs after 24 h is shown in Figure 2
. In this figure the x-axis was calibrated for LPS
together with the number of H44/76 bacteria that contained an
equivalent amount of LPS, based on the above-presented TSDS-PAGE
results. Therefore, the effect of all three stimuli could be compared
quantitatively.
 View larger version (15K): [in a new window] |
Figure 2. Production of TNF- (left panel), iL-1ß (middle panel), and IFN-
(right panel) after 24 h by human PBMCs stimulated with different
concentrations of H44/76 LPS (•), wild-type H44/76 meningococci
( ), and LPS-deficient H44/76[pLAK33] meningococci ( ). Values on
the x-axis are calibrated for H44/76 LPS and the number of
H44/76 bacteria that contain the same amount of LPS (1 ng of LPS 7x105 bacteria). Mean values ± SE
are presented (n=5).
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and IL-1ß than equivalent amounts of isolated LPS
(P<0.05). Below these concentrations, LPS was a more potent
inductor of TNF- and IL-1ß production. LPS-deficient pLAK33
meningococci were able to induce substantial amounts of TNF-,
IL-1ß, and IFN- production. For TNF- and IL-1ß, approximately
10-fold-higher amounts of LPS-deficient bacteria were required to
induce the same level of cytokine production as that in wild-type
bacteria. Of interest, significant IFN- production occurred after
stimulation with both the wild-type and the LPS-deficient meningococci,
but only minute amounts of IFN- were produced after stimulation with
isolated LPS (P<0.05). Experiments (n=8)
performed with 4 h of incubation showed a similar pattern for
TNF- and IL-1ß (data not shown).
To assess whether the IFN- production induced by LPS-containing or
LPS-deficient meningococci is secondary to the TNF- or IL-1ß
production, a series of induction experiments with 6 x
108/mL of H44/76 and pLAK33 bacteria was performed with
cells from 30 donors. In these experiments, the TNF- and IL-1ß
production appeared to be correlated (r=0.061
(P<0.01) and r=0.70 (P<0.001) for
wild-type and LPS-deficient meningococci, respectively). However,
IFN- production did not correlate with the TNF- or IL-1ß
production values for r between -0.12 and 0.22
(P=not significant). In addition, it could be seen that
IFN- production showed a striking interindividual variety [range,
56–7,800 pg/mL (median, 555 pg/mL) and 40–6800 pg/mL (median, 660
pg/mL) for H44/76 and pLAK33 bacteria, respectively]. Thus, IFN- is
probably not produced in response to TNF- or IL-1ß, and the
individual response in IFN- production after stimulation with
meningococci differs considerably.
The time course for TNF- and IL-1ß production by PBMCs showed a
similar pattern for meningococcal LPS and both meningococcal strains.
In brief, TNF- became detectable after 2 h, reached a maximum
after 8 h, and declined thereafter whereas IL-1ß was detectable
after 4 h and reached a plateau phase after 12 h (data not
shown).
The relative contribution of non-LPS structures to the total induction
of TNF- and IL-1ß induction by N. meningitidis was
assessed in a series of combination experiments. In these experiments,
meningococci were supplemented with approximately the same amount of
LPS as present in the wild-type strain. Figure 3
shows the results of this experiment for IL-1ß after 24 h
of incubation. It can be seen that IL-1ß production induced by
LPS-deficient bacteria was approximately half of that induced by
LPS-containing bacteria, whereas after addition of LPS, the IL-1ß
induction by LPS-deficient bacteria was restored to the level of
LPS-containing bacteria. Results for TNF- and for experiments
(n=5) with 4 h of incubation showed a similar pattern
(data not shown). This additive effect of LPS to the non-LPS-induced
cytokine synthesis suggests that both stimuli may use different
pathways for the induction of TNF- and IL-1ß.
 View larger version (48K): [in a new window] |
Figure 3. IL-1ß production after 24 h by human PBMCs stimulated with
6 x 106 or 6 x 108/mL of wild-type
H44/76 meningococci (open bars) (n=25), the same amounts of
LPS-deficient H44/76[pLAK33] meningococci (light-gray bars)
(n=25), or the same amounts of LPS-deficient pLAK33
meningococci substituted with equivalent amounts of LPS (0.0085 and
0.85 µg/mL, respectively) as present in wild-type H44/76 meningococci
(dark-gray bars) (n=5). Mean values are presented ±
SE. **, P < .01 compared with wild-type
meningococci as well as LPS-deficient meningococci substituted with
LPS.
|
and for experiments (n=5) with 4 h of
incubation showed a similar pattern (data not shown). Taken together
these results indicate that the outer membrane components present in
OMCs like PorA, PorB, RmpM, or OpA make a minimal contribution to
cytokine induction.
 View larger version (14K): [in a new window] |
Figure 4. Production of IL-1ß after 24 h by human PBMCs stimulated with
different concentrations of OMCs isolated from the wild-type H44/76
meningococci () containing 10–20% LPS and from mutant
H44/76[pLAK33] meningococci () devoid of LPS. OMC concentration is
expressed as protein content in micrograms per milliliter. Mean values
(n=5) are presented ± SE.
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View this table: [in a new window] |
Table 1. Production of mTNF- and mIL-1 after 24 h by Murine
Peritoneal Macrophages Stimulated with Wild-Type H44/76
Meningococci and LPS-Deficient H44/76[pLAK33] Meningococci
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|
View this table: [in a new window] |
Table 2. Mortality at 24 h (%) in Mice Pretreated with Galactosamine after
i.v. Injection of Heat-Killed N. meningitidis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-1ß production, that non-LPS components
induced IFN-, and that non-LPS parts could provoke disease. Based on
TSDS-PAGE analysis of LPS in proteinase K-treated cell lysates of
N. meningitidis H44/76, a quantitative comparison between
the cytokine-inducing capacity of LPS and that of LPS-containing
bacteria became possible. It was shown that concentrations of
>107/mL of bacteria were more potent inductors of cytokine
synthesis than equivalent amounts of isolated LPS and that cytokine
induction by non-LPS components was additive to that by LPS.
Furthermore, it was demonstrated that the non-LPS components
responsible for the cytokine induction do not reside in
sarcosyl-extracted outer membrane complexes.
Non-LPS components of bacteria can induce cytokine production
[7
], as has been shown by experiments with gram-positive
bacteria [27
28
29
30
31
] and with various isolated elements of
these bacteria like peptidoglycan [32
33
34
], lipopeptides
[28
], lipoproteins [28
], lipoteichoic
acid [35
], and capsular polysaccharides
[36
]. However, so far studies trying to assess
quantitatively the contribution of these non-LPS structures to cytokine
induction by gram-negative bacteria were hampered by the inevitable
copresence of LPS, outflanking the cytokine induction by non-LPS
structures. To circumvent this problem we used a meningococcal mutant
that is entirely deficient of LPS. With this strain we demonstrated
that approximately half the amount of TNF- and IL-1ß induced by
meningococci in human PBMCs or murine peritoneal macrophages was
elicited by non-LPS structures. Similarly, LPS-deficient meningococci
could cause lethal disease in galactosamine-pretreated mice, albeit the
dosages of LPS-deficient meningococci required to induce mortality were
approximately 100-fold higher than for wild-type meningococci.
TNF-, IL-1ß, and IFN- are pivotal mediators in the pathogenesis
of Gram-negative septic shock. After infusion of LPS in human
volunteers or primates, TNF- and IL-1ß appear within 1 to 2 h
[37
], but no IFN- seems to appear
[38
]. However, after the infusion of whole bacteria,
IFN- is induced and can be detected in the circulatory system after
6–8 h [38
, 39
]. During meningococcal
infections, IFN- is increased in plasma or cerebrospinal fluid and
correlates with the severity of disease [40
,
41
]. In our in vitro study, meningococcal LPS stimulated
only minimal IFN- production, whereas both LPS-containing and
LPS-deficient bacteria induced significant amounts of IFN-. In
addition, IFN- was not produced in response to TNF- or IL-1ß,
which indicates that non-LPS components of meningococci were primarily
responsible for IFN- induction. Because the primary source of
IFN- is the lymphocyte and marked differences occur between
individuals, it is tempting to speculate that certain non-LPS
components of meningococci can act as superantigens.
In this study we used the widely employed KDO assay to determine the amount of LPS in the H44/76 LPS preparation [22 , 42 , 43 ]. With this method (0.8/0.92) x 100% (i.e., 85%) of the LPS was detected. Limitations of this assay are conversion of a fraction of KDO during acid hydrolysis to entities inert to the thiobarbituric acid reaction and incomplete hydrolysis, both leading to an underestimation of the amount of KDO. On the other hand, contaminants in the LPS may coreact in the assay, which leads to overestimation of the amount of LPS [44 , 45 ]. The relatively accurate yield of KDO in the present study showed that these limitations of the KDO assay are likely to be of minor importance for determination of H44/76 LPS.
By TSDS-PAGE analysis of LPS in lysates of N. meningitidis H44/76, the amount of LPS in H44/76 bacteria was determined. It appears that 7 x 105 bacteria corresponded to approximately 1 ng of LPS, which fits rather well with the results obtained by KDO analysis in H44/76 and pLAK33 bacteria. This estimate is in good accordance with reported estimates of 1 ng of LPS for 105 bacteria with Escherichia coli [1 , 46 47 48 49 50 ], taking into account that the MW of meningococcal LPS is 2- to 10-fold lower than that of E. coli LPS. In addition, our estimate compares fairly well to clinical data of Brandtzaeg et al. [51 ], Mariani-Kurkdjian et al. [52 ], and Bingen et al. [53 ], who detected LPS values up to 500 ng/mL and bacterial numbers up to 5 x 108 CFU/mL in cerebrospinal fluid during meningococcal meningitis.
Based on the estimate that 7 x 105 bacteria
correspond to 1 ng of LPS, we could compare the cytokine-inducing
potency of isolated LPS with that of meningococci containing the same
amount of LPS. It was found that at concentrations below
107 bacteria/mL or equivalent amounts of LPS, LPS induced
more TNF- and IL-1ß, but at higher concentrations, complete
meningococci were more potent. Because at these higher concentrations
the TNF-- and IL-1ß-inducing capacities of LPS-deficient
meningococci increased in a parallel fashion, the higher activity of
bacteria at these higher concentrations is likely to have been caused
by the non-LPS components of the bacterium.
Fulminant meningococcal sepsis is characterized by high plasma concentrations of endotoxin that range from 0.75 to 170 ng/mL [8 , 10 , 54 55 56 ]. Based on our estimation of 1 ng of LPS per 7 x 105 bacteria, the reported range of endotoxin concentrations corresponds to 5 x 105 to 1.2 x 108 bacteria/mL. We speculate that strategies designed to block LPS-induced cytokine synthesis alone are of limited value, because at this degree of bacteremia, a substantial part of the proinflammatory cytokine response is elicited by non-LPS parts of the meningococcus [56a ].
Because several outer membrane proteins of N. meningitidis are known to interact with human cell receptors [57 , 58 ], we examined whether sarcosyl-extracted outermembrane complexes are able to induce cytokine production. LPS-deficient OMCs primarily composed of the major outer membrane proteins PorA, PorB, RmpM, and OpA did not induce cytokines. Thus, other meningococcal components not retained after sarcosyl extraction, for instance certain lipoproteins, chromosomal DNA, the polysaccharide capsule [59 ], or the peptidoglycan cell wall [32 33 34 ], must have been responsible for the observed cytokine induction by the LPS-deficient mutant. Further research is needed to identify which of the non-LPS components are responsible for cytokine induction and to quantify their relative contribution to the pathogenesis of invasive meningococcal disease.
Received July 26, 2000; revised March 31, 2001; accepted April 5, 2001.
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