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Regular Article |
From the Department of Pathology, University of Michigan, Ann Arbor, Michigan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several groups of chemokines have been identified based on the relative position of two conserved cysteine residuals (C, CXC, CX3C).3 CXC chemokines that incorporate an ELR motif (ELR+) near the N-terminus are particularly potent inducers of neutrophil chemotaxis. In humans, seven ELR(+)CXC chemokines have been identified, of which interleukin (IL)-8 seems to be the most important neutrophil attractant and activator during acute inflammation.3-5 Studies of inflammation necessarily involve animal models, and murine models have been used extensively in this context. Nevertheless, no structural homologue for IL-8 has been identified in mice. Although several murine ELR(+)CXC chemokines have been identified to date [ie, KC, macrophage inflammatory protein (MIP-2), and LIX], they are structurally homologous to human chemokines other than IL-8.6
During an inflammatory event, concentrations of cytokines typically increase more at the local site of inflammation compared to systemic levels.7,8 At a compartmental level (eg, peritoneum), this has not necessarily been true for murine chemokines. In a murine cecal ligation and puncture model, Walley and colleagues9 found significantly elevated, but approximately equal concentrations of peritoneal and serum MIP-2. In their studies of cecal ligation and puncture, Ebong and colleagues10 also reported approximately equal increases in peritoneal lavage and plasma MIP-2, but found a >10-fold higher concentration of plasma KC compared with peritoneal lavage. These latter kinetics studies illustrated how the complexities of sepsis can decouple any clear correlations between chemokine levels and neutrophil recruitment.
In the present study we examined the in vivo relationship between murine chemokines and neutrophil trafficking. To avoid the complexities inherent in a sepsis model, we used a thioglycollate model of inflammation. It has long been recognized that intraperitoneal injections of thioglycollate in mice will induce rapid and abundant recruitment of neutrophils into this compartment without inducing degranulation.11 Consequently, this represents a relatively simple model that, when combined with neutralizing antibodies for KC and MIP-2, offers a unique opportunity to gain insight into the relative contribution of individual murine chemokines to neutrophil trafficking. We also examined how KC and MIP-2 might contribute to expression of other inflammatory mediators.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ELISAs
We developed sandwich ELISAs for KC and MIP-2 using our polyclonal antibodies. IgG was purified from rabbit sera using protein A columns (Pierce Chemical Co., Rockford, IL) and these polyclonal antibodies were used for both capture and detection. Secondary antibodies were conjugated to biotin (Pierce). Streptavidin-conjugated horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) was used with 3,3',5,5' tetramethylbenzidine substrate (Genzyme, San Carlos, CA) for colorimetric detection and quantification. We used commercially available recombinant KC and MIP-2 for our standard curves (R&D Systems, Minneapolis, MN). This provided an independent verification of both the sensitivity and specificity of our ELISAs. Both KC and MIP-2 ELISAs were sensitive (<20 pg/ml) and showed no cross-reaction over the range of our standard curves (2 pg/ml to 20 ng/ml). Both KC and MIP-2 sandwich ELISAs were partially suppressed in the presence of plasma or serum. Consequently, when assaying plasma for these chemokines, the standard curve always contained an equivalent dilution of normal mouse plasma.
Tumor Necrosis Factor (TNF)-
and IL-6 Assays
TNF- bioactivity was assessed using the WEHI 164 subclone 13
bioassay13
as previously described.14,15
IL-6
was quantified using a B9 cell proliferation assay16
following the methods of Wollenberg and colleagues.17
We
routinely detected <3 pg/ml of biologically active TNF- and IL-6
using these bioassays in conjunction with recombinant protein standards
(TNF- from Cetus Immune Corp., Emeryville, CA; IL-6 from
Preprotech, Rocky Hill, NJ).
In Vivo Neutralization Experiments
Normal BALB/c mice (17 to 19 g; Harlan, Indianapolis, IN) were passively immunized by subcutaneous injection of anti-KC (goat), or anti-MIP-2 (rabbit) or both antibodies or control serum (goat and rabbit). All serum was heat inactivated (56°C, 30 minutes) and filter-sterilized before use. Injections were composed of 333 µl of antiserum, 333 µl of normal saline, and 333 µl of normal serum from goat (if anti-KC) or rabbit (if anti-MIP-2). Normal serum was not added when both antibodies were used. After 2 hours, immunized mice were lightly anesthetized with methoxyflurane (Shering-Plough, Union, NJ) and given an intraperitoneal injection of 2.0 ml of sterile thioglycollate (0.3% or 3.0%; Difco, Detroit, MI). Four hours later mice were anesthetized (ketamine, 1.7 mg/mouse and xylazine, 1.3 mg/mouse) and exsanguinated via retro-orbital plexus bleed into heparinized tubes. Plasma was stored at -20°C for later analysis. Mice were immediately euthanized via cervical dislocation and the peritoneal cavity was opened aseptically. Any residual thioglycollate was collected or if none was present, the cavity was flushed with 1.0 ml of ice-cold Hanks’ buffered salt solution (no Mg+2 or Ca+2; Life Technologies, Gaithersburg, MD). Cells from this initial wash were pelleted and the peritoneal supernatant stored at -20°C after recording the total volume recovered. The peritoneal cavity was then washed with 20 ml of Hanks’ buffered salt solution and all cells collected from the peritoneal cavity were resuspended together. Cytospins were prepared for differential counts and total leukocytes were quantified with a Coulter counter (Coulter Corp., Miami, FL). KC and MIP-2 were quantified for the plasma and peritoneal samples, and the latter was expressed as the total amount of chemokine recovered to reflect differences in volume recovery between animals. The heart was perfused with 2 to 5 ml of normal saline and the right lung removed to quantify the level of myeloperoxidase present in the tissue.18,19 This latter assay is an indicator of the relative number of neutrophils that are sequestered in the pulmonary capillary beds and serves as an indicator of neutrophil adhesion and rigidity.20
Statistical Analyses
Comparisons between treatments and controls were made using Wilcoxon signed-rank tests. We used paired Wilcoxon signed-rank tests or Mann-Whitney U tests for other two-way comparisons. Pearson correlations were reported for tests of association. All statistical calculations were made using NCSS 97,21 or SigmaStat (SPSS, Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal mice had few neutrophils within the peritoneum and IL-6 was
not detectable (Table 1)
. We evaluated
the peritoneal response to 0.3% and 3% thioglycollate and found a
dose-dependent increase in the peritoneal concentration of IL-6 and the
number of recruited neutrophils (Table 1)
. In this limited study, a
10-fold increase in the thioglycollate concentration caused a 10-fold
increase in the IL-6 levels, and a 10-fold increase in neutrophil
recruitment.
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We next examined the local and systemic production of chemokines
in response to the thioglycollate injections. To better understand the
contributions of the individual chemokines to this recruitment, we also
passively immunized mice against the murine chemokines KC and MIP-2. In
normal mice, plasma and peritoneal levels of the murine chemokines are
generally below detection limits (data not shown). Four hours after
intraperitoneal injection of 3% thioglycollate, there was a
substantial increase in both the peritoneal and plasma levels of the
murine chemokines (Figure 1)
. Plasma and
peritoneal levels of IL-6 were also increased, indicating both a
systemic and local inflammatory response. Antibody inhibition of KC
reduced both plasma and peritoneal levels of KC. In a similar manner,
antibody inhibition of MIP-2 reduced MIP-2 concentrations both in the
plasma and the peritoneum (Figure 1)
. As we have previously reported,
the levels of KC are always greater than the levels of
MIP-2.10
A somewhat surprising finding was that inhibition
of both MIP-2 and KC caused a significant reduction in the plasma and
peritoneal concentrations of IL-6. There was no TNF- detected in any
samples from animals treated with 3.0% thioglycollate.
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. Blockade of KC and
MIP-2 reduced neutrophil counts 80% and 76%, respectively (Figure 2)
.
Immunization against both chemokines reduced neutrophil recruitment by
93%. Immunizing against MIP-2 or against both KC and MIP-2
significantly reduced pulmonary neutrophil sequestration (data not
shown).
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. In this model, however, inhibition of
KC alone resulted in a significant reduction in both the plasma and
peritoneal levels of MIP-2. This inhibition is not because of
nonspecific cross-reactivity between the anti-KC antibody and MIP-2
protein (unpublished observations). Additionally, blockade of KC
resulted in significant reductions in the levels of IL-6. Again, no
biologically active TNF- was detectable in the specimens.
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. After passive immunization against
KC, 0.3% thioglycollate treatment resulted in a sevenfold increase in
the number of peritoneal neutrophils over control antibody animals.
Antibody inhibition of MIP-2 also increased peritoneal neutrophil
recruitment, but the results were not significant. The KC antibody
inhibition also caused a significant reduction in neutrophil
sequestration within the lung, as indicated by reduced pulmonary
myeloperoxidase (data not shown).
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, KC seems to be the more important chemokine for the
recruitment of neutrophils, and therefore we quantitated the ratio of
peritoneal to plasma KC after either 0.3% or 3% thioglycollate
challenge. This was done to test the hypothesis that the ratio of local
to systemic chemokines dictates the biological response. As indicated
in Table 2
, the changes in the ratio of
peritoneal to plasma chemokines observed with antibody inhibition
provided an explanation for the observations of the neutrophil
recruitment. After 3% thioglycollate injection, the ratio of
peritoneal to plasma KC was 0.22. This ratio was reduced 50% with
antibody inhibition, and the number of recruited neutrophils was
reduced nearly fourfold. In contrast, for the 0.3% challenge the
peritoneal to plasma ratio of KC was increased 36-fold with antibody
inhibition, and the local recruitment of neutrophils was increased
sevenfold. Antibody inhibition of MIP-2 reduced neutrophil recruitment
for animals receiving 3% thioglycollate (23 x
105
to 6.8 x 105). In
this case we again found a reduction in the peritoneal to systemic
concentrations of MIP-2 (6.4 to 1.0, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite these studies and the work of Ebong and colleagues,10,14 the precise individual chemokine contribution to the inflammatory process is not clear. Both KC and MIP-2 have been shown to elicit neutrophil chemotaxis in vitro and in vivo27-29 and both proteins bind equally well to the murine homologue for IL-8 receptor B.29,30 Both KC and MIP-2 up-regulate Mac-1 on neutrophils; an important adhesion molecule involved in neutrophil chemotaxis.28,29 In this study, inhibition of MIP-2 (3% challenge) or KC (0.3% challenge) reduced myeloperoxidase significantly; a result that suggests both chemokines may be involved in neutrophil stiffening subsequent to extravascular transmigration.20 The high degree of functional redundancy between KC and MIP-2 may be exploited as a means for organ- and cell-specific regulation of leukocytes. For instance, tissue-specific differences were found with an endotoxin model of inflammation. KC was expressed highest in lung, heart, and liver whereas MIP-2 was mostly expressed in lung and to a lesser extent heart tissue.6 KC also is constitutively expressed and may serve an important role in maintaining neutrophils outside the vascular compartment.6,12,30 For our model of more severe peritonitis (3% thioglycollate), both KC and MIP-2 seemed to play significant roles in the inflammatory process, probably via redundant and distinct mechanisms.
Our less severe model of peritonitis (0.3% thioglycollate) demonstrated a more significant relationship between KC and neutrophil recruitment when one calculates the peritoneal:plasma ratio. This model, although eliciting 10-fold fewer neutrophils (compared to 3% model) still induced high local and systemic levels of KC, but reduced levels of MIP-2 and IL-6. In animals treated with control anti-sera, the high level of systemic KC relative to peritoneal KC may have disrupted the chemotactic gradient into the peritoneum. When we immunized against KC, plasma levels of KC plummeted while there was less reduction in the peritoneal KC concentration. Despite the decreased levels of the chemoattractant there still seemed to be sufficient local KC (>1 ng) to attract circulating neutrophils to the peritoneum. Consequently, it is apparent that the easily induced and very high levels of systemic KC can reverse the chemotactic gradient or otherwise interfere with chemotaxis and thus serve as an anti-inflammatory mediator in the face of minor irritants. Others have shown that administration of IL-8 will reduce neutrophil adhesion in vitro31 and in vivo.32-34 Ley and colleagues33 concluded that high systemic levels of IL-8 inhibited transmigration of neutrophils by interfering with L-selectin-independent adhesion. Simonet and colleagues34 also concluded that high systemic levels of IL-8 could interfere with adhesion, although they also addressed the possibility of a reversal of chemotactic gradients. The results of our 0.3% thioglycollate model seem to mimic the anti-inflammatory actions of the former IL-8 experiments.
A surprising finding in these studies is the observation that injection of a lower concentration of thioglycollate (0.3%) resulted in higher plasma levels of IL-6 relative to the injection of 3% thioglycollate. The lower concentration of thioglycollate did result in significantly fewer neutrophils recruited into the peritoneum, and reduced peritoneal levels of IL-6 and MIP-2. We thus have the paradoxical result in which less inflammation apparently induced higher levels of plasma IL-6. Although we do not have a definitive answer for this observation, it may be related to the timing of sampling. In a sepsis model it has been shown that the more severe sepsis results in peak plasma levels of IL-6 at a later time point.10 Because all of the plasma samples were obtained at 4 hours after injection, it is possible that the higher concentration of thioglycollate would have induced greater concentrations of plasma IL-6 at later time points.
Several investigators have documented that increased local levels of chemokines are associated with increased recruitment of inflammatory cells. In animal models, higher local levels of chemokines increase neutrophil recruitment. This has been observed after a challenge with staphylococcal superantigens35 or injection of recombinant tumor necrosis factor which causes a subsequent increase in local chemokine production.36 In both of these studies, antibody inhibition of the chemokines reduced local neutrophil recruitment. In a streptococcal arthritis model, high local levels were found relative to systemic levels.37 Chemokines have also been shown to be important in recruitment of neutrophils into infected urinary bladders.38 In an interesting model of Pseudomonas infection, low doses resulted in only local levels of chemokines but not systemic levels.39 In humans, increased levels of CXC chemokines in the lung40 or cerebrospinal fluid41 correlated with increased numbers of neutrophils.
Our results are similar to a previous study, which documented that the gradient of local to systemic chemokines was closely related to recruitment of neutrophils. For this model, Blackwell and colleagues42 manipulated the pulmonary chemokine levels by intratracheal injection of endotoxin, whereas systemic levels were altered by intraperitoneal levels of endotoxin. By increasing the plasma levels of the chemokines relative to the pulmonary levels, they were able to dramatically decrease the influx of neutrophils. Our data show a similar pattern in which the ratio of the local to the systemic chemokine concentrations, rather than the absolute local chemokine concentration, dictates the recruitment of neutrophils. Therefore, knowing only the local levels of chemokines, or only the plasma levels, provides only half of the data needed for the equation to predict inflammatory cell recruitment.
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Footnotes |
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Supported by National Institutes of Health grants GM 44918 and GM 50401. Part of this work was recognized for a 1999 Postdoctoral Merit Award (to D. R. C.) by the American Society for Investigative Pathology.
Current address of D. R. Call: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-4070
Accepted for publication October 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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