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INFLAMMATION AND CYTOKINES
production in neutropenia
Department of Surgery, Division of Surgical Research, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island
Submitted 14 May 2004 ; accepted in final form 23 September 2004
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ABSTRACT |
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concentration after LPS injection and to enhance LPS-induced mortality. Experiments reported here demonstrate that the 15-fold higher plasma TNF- concentration elicited by LPS in neutropenic vs. nonneutropenic unanesthetized mice correlated with increased hepatic and splenic, but not pulmonary, TNF- mRNA. Core 2 
-1,6-N-acetylglucosaminyltransferase-null and CD18-deficient mice also exhibited exaggerated plasma TNF- responses to LPS injection. Findings suggest that extravasated neutrophils inhibit systemic TNF- production and that they do so through organ-selective mechanisms involving CD18 integrin and selectin binding. inflammation; neutrophil; lipopolysaccharide; CD18; Core 2 oligosaccharide
(1). To explain this exaggerated inflammatory response in neutropenia, it was proposed that neutrophil-depleted animals have a relative lack of circulating neutrophil-derived TNF--neutralizing TNF receptor p75 (19). Alternatively, an impairment in TNF- clearance in the absence of neutrophils was implicated as a causative mechanism (8).
Experiments reported here examined the impact of neutropenia on the expression of TNF- mRNA in liver, spleen, and lung of neutropenic and control animals given endotoxin. Results demonstrate that TNF- mRNA is higher in the liver and spleen, but not in the lung, of neutropenic mice than in controls after endotoxin injection. Moreover, results obtained from Core 2 -1,6-N-acetylglucosaminyltransferase (Core 2)-null and CD18-deficient animals demonstrate that cell adhesion molecules play a role in the regulation of plasma TNF- in endotoxemia. These findings indicate that inhibition of LPS-induced TNF- production derives from extravasated neutrophils and that regulation occurs differentially among organs.
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METHODS |
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Induction of neutropenia. Monoclonal anti-Gr-1 antibody (RB6.8C5 hybridoma originally produced by R. L. Coffman, DNAX Research Institute, Palo Alto, CA) was produced under serum-free conditions with the use of a bioreactor (Cell Pharm Micro Mouse, UniSyn Technologies, Tustin, CA). Mice were rendered neutropenic by a single intraperitoneal injection of 0.5 mg of anti-Gr-1 antibody 3 days before LPS injection. Control animals were injected intraperitoneally with an equal volume of normal saline. Neutropenia was documented by differential blood leukocyte count of Hema-3 (Biochemical Sciences, Swedesboro, NJ)-stained smears taken from the lateral tail vein before LPS injection. Preliminary experiments demonstrated that neutropenia persists for at least 4 days after antibody injection.
LPS injection.
Mice were injected intraperitoneally with 1 mg/kg LPS (E. coli serotype 055:B5, Sigma, St. Louis, MO). At specified times after LPS injection, animals were euthanized by CO2 asphyxiation. Heparinized blood obtained from the inferior vena cava was immediately centrifuged at 800 g for 10 min, and plasma was frozen at –70°C until analyzed for TNF-. Additional animals were euthanized 30 min after LPS injection; liver, spleen, and lung tissues were obtained for real-time PCR analysis.
TNF- assay.
TNF- was assayed by ELISA with antibody pairs (BD Biosciences, San Diego, CA). The recovery of recombinant murine TNF- added to a 1:20 dilution of plasma was 93%. TNF- bioactivity was determined by lysis of actinomycin D (Sigma)-treated L929 murine fibroblasts (NCTC clone 929, American Type Culture Collection) (11). One unit was defined as the quantity of TNF- resulting in 50% lysis of L929 cells.
Quantitative PCR.
Total RNA was obtained from liver, spleen, and lung. Isolated tissues were immediately immersed into RNAlater (Qiagen, Valencia, CA) and stored at –20°C. RNA extraction was performed by using RNeasy Mini kit (Qiagen). RNA (1 µg) was reverse transcribed with the use of murine reverse transcriptase (Amersham, Piscataway, NJ). For each reaction set, one RNA sample was run without murine reverse transcriptase to provide a negative control in subsequent PCRs. PCR primers for TNF- were as follows: 5'-CACGCTCTTCTGTCTACTGA-3' (forward) and 5'-CACTTGGTGGTTTGCTACGA-3' (reverse) (Integrated DNA Technologies, Coralville, IA). Primers for -actin were 5'-TGTGATGGTGGGAATGGGTCAG-3' (forward) and 5'-TTTGATGTCACGCACGATTTCC-3' (reverse) (Stratagene, Cedar Creek, TX). Real-time PCR was performed by using Stratagene Mx4000 quantitative PCR system according to the manufacturer's instructions. Reactions were optimized and performed with brilliant SYBR green quantitative PCR core reagent kit (Stratagene). Each PCR amplification was performed in triplicate wells, using the following conditions for TNF- gene: 10 min at 95°C followed by 40 cycles of 30 s at 95°C, 1 min at 55°C, 30 s at 72°C, and final extension for 10 min at 72°C. For -actin, conditions were as follows: 10 min at 95°C followed by 40 cycles of 30 s at 95°C, 1 min at 63°C, and 45 s at 72°C with final extension 10 min at 72°C. Results are reported as TNF--to--actin copy ratio.
Histology. Formalin-fixed samples of tongue, skin, lung, liver, and spleen were obtained from neutropenic and control animals 3 days after injection of anti-Gr-1 antibody or saline. Tissues were embedded in paraffin, sectioned, and stained with toluidine blue (Sigma) (5). The number of mast cells in 10 high-power fields was determined in tongue, dermis, lung parenchyma, liver, and spleen.
Data presentation. Results shown are means ± SE or SD, as indicated in the text. Differences among groups were tested by two-factor ANOVA with Newman-Keuls, or by Mann-Whitney U-test, as appropriate.
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RESULTS |
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response following endotoxin administration.
Intraperitoneal injection of 0.5 mg of monoclonal antibody directed against the neutrophil surface antigen Gr-1 reduced neutrophils to 2.2 ± 1.8% of circulating blood leukocytes vs. 10.9 ± 4.1% in controls (mean ± SD, P < 0.01, Mann-Whitney U-test). Anti-Gr-1 antibody did not alter circulating lymphocyte or circulating monocyte counts (data not shown). Tissue mast cell content in neutropenic animals did not differ from controls (Table 1).
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concentrations in control and neutropenic animals following the injection of LPS. The peak plasma TNF- concentration was 15-fold higher in neutropenic mice than in controls. LPS injection in control animals resulted in peak plasma TNF- concentration of 4.9 ± 1.7 ng/ml at 60 min, compared with 17 ± 1 pg/ml before LPS injection (P < 0.05, two-factor ANOVA, Newman-Keuls). Peak plasma TNF- concentration following LPS injection of neutropenic animals was 76.6 ± 16.1 ng/ml at 90 min compared with 21 ± 3 pg/ml before LPS injection (P < 0.05, two-factor ANOVA, Newman-Keuls). TNF- bioassay of plasma obtained 90 min after intraperitoneal injection of LPS confirmed the exaggerated TNF- response in neutropenic animals (4,898 ± 1,483 U/ml in neutropenic animals vs. 218 ± 151 U/ml in controls, mean ± SD, P < 0.01 by Mann-Whitney U-test).
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mRNA after endotoxin administration is higher in the liver and spleen of neutropenic animals than in controls.
To assess transcriptional changes preceding and determining increased plasma TNF- concentrations, the liver, spleen, and lungs were harvested from control and neutropenic mice 30 min after LPS injection and analyzed for TNF- mRNA by quantitative PCR. Results presented in Table 2 demonstrate that livers and spleens of endotoxemic neutropenic animals contained more TNF- mRNA than those from endotoxemic controls. Lung TNF- mRNA was not different in neutropenic and control mice.
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response to endotoxin.
Mice deficient for selectin ligands (Core 2-null) or -integrins (CD18-deficient) were injected with LPS, and plasma TNF- concentrations were determined 90 min later. As illustrated in Fig. 2, both adhesion molecule-deficient animals showed enhanced plasma TNF- responses to LPS compared with wild-type controls.
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DISCUSSION |
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production and the appearance of TNF- in plasma. It has been proposed from studies involving partial hepatectomy (12) and the selective depletion of Kupffer cells (16) that these cells are a source of TNF- in endotoxemia. However, murine mast cells contain preformed TNF- (6), which is released in vitro in response to LPS (13). It was considered that differences in mast cell number or mast cell activation could account for the exaggerated plasma TNF- response seen in endotoxemic neutropenic animals. Table 1 demonstrates that tissue mast cell counts did not differ in neutropenic animals compared with controls. Also, mast cells do not contribute to the rapid appearance of TNF- in the serum of mice following intraperitoneal injection of LPS (4). Thus it is unlikely that mast cells are a source of LPS-induced TNF- in neutropenic mice.
TNF- and neutrophils have been shown to be involved in determining liver injury following endotoxin administration (8). In studies of the role of neutrophils in postendotoxemic liver injury, Hewett et al. (8) first described a marked increase in circulating TNF- in endotoxin-injected neutropenic animals. The exaggerated plasma TNF- response in neutropenia is not restricted to endotoxemia because it has also been shown to occur in experimental C. albicans infections (20).
Results reported here confirm and extend the findings of Hewett et al. (8) and others and provide evidence for a transcriptional mechanism for the exaggerated production of TNF- by demonstrating increased cytokine mRNA in the livers and spleens, but not in the lungs, of endotoxemic neutropenic mice. Differences among organs may have resulted from differences in the relative macrophage content or differences in the macrophage phenotype in the liver, spleen, and lung. Alternatively, the differences among organs might have been caused by the intraperitoneal route of LPS administration.
Previous proposals to explain the increased TNF- in the blood of endotoxemic neutropenic animals have focused on the neutralization of TNF- bioactivity by soluble TNF- receptors and the increased catabolism of TNF-. In this connection, it was shown that mice made neutropenic with cyclophosphamide and injected with endotoxin have lower circulating soluble TNF receptor p75 than controls (19). A reduction in circulating TNF- receptors known to bind and neutralize TNF- could explain the changes in TNF- bioactivity in the plasma, which were described by Steinshamn et al. (19) and are confirmed in the present study, but would not explain the changes in immunoreactive TNF- that are reported in Fig. 1. Hewett et al. (8) proposed that decreased clearance of TNF- could contribute to the increased plasma cytokine concentration in neutropenic animals but did not provide evidence in support of that hypothesis. Finally, it has been shown that neutrophils release proteolytic enzymes capable of degradation of TNF- in culture (21). The relevance of this mechanism to the biology of TNF- in vivo has not been studied. The present finding that neutropenia determines organ-selective increases in TNF- mRNA suggests that the increase in plasma TNF- in neutropenic endotoxemic animals stems from increased cytokine synthesis in the liver and the spleen.
The present observation that neutrophil depletion resulted in increased postendotoxin plasma TNF- concentration and hepatic and splenic TNF- mRNA suggested the hypothesis that neutrophils actively suppress the production of TNF- in endotoxemia. Moreover, present results suggested that neutrophil arrival into different organs in endotoxemia might serve to blunt the production of TNF-. In this connection, it is known that endotoxin administration leads to the rapid sequestration of neutrophils in the liver, spleen, and lungs (17). By extension of the hypothesis, blocking neutrophil infiltration of solid organs following endotoxin administration should also result in exaggerated TNF- production. To test this hypothesis, experiments were performed using Core 2-null and CD18-deficient animals.
Core 2 oligosaccharides are the products of the Golgi enzyme Core 2--1,6-N-acetylglucosaminyltransferase (3). These oligosaccharides provide the scaffold for the production of carbohydrate adhesion ligands on the surface of leukocytes that bind the selectin family of adhesion molecules. The role of selectins in modulating neutrophil entry into the solid organs remains controversial. Wong et al. (23) reported that neither E-selectin/P-selectin-deficient animals nor the same animals injected with an anti-L-selectin antibody exhibited changes in leukocyte adhesion in the liver sinusoids during perfusion with formyl-methionyl-leucyl-phenylalanine. Furthermore, neutrophil infiltration of the livers of E-selectin/P-selectin-deficient mice after LPS administration did not differ from that shown in wild-type mice (23). In contrast, studies in rats reported by Shi et al. (18) indicated that blocking of P-selectin with antibody or with injection of the P-selectin antagonist low-molecular-weight heparin reduced the arrival of neutrophils to the liver in endotoxemia.
CD18 is the -chain of leukocyte CD11/CD18 integrins that regulate multiple cell functions, including adherence to endothelium, extravasation and migration into solid organs, and phagocytosis (2). Neutrophil recruitment into the liver has been shown to require integrin activation. Specifically, it has been shown that antibodies against CD11b or CD18 prevent the infiltration of neutrophils into the liver following galactosamine/endotoxin administration in mice (9).
Results shown in Fig. 2 demonstrate exaggerated plasma TNF- responses to endotoxin in Core 2-null and CD18-deficient mice. Although the cytokine plasma concentration induced by LPS injection was less in the adhesion molecule-deficient mice than in the neutropenic mice, these findings indicate that interference with either integrin-dependent adhesion or selectin-mediated cell rolling results in excess plasma TNF- accumulation in the plasma after LPS injection.
Observations just discussed are congruent with the hypothesis that neutrophils arriving into solid organs as a result of LPS administration moderate or suppress the production of TNF-. A corollary to these findings is that neutrophils have an anti-inflammatory role, inhibiting activation of and cytokine release from other cells. This hypothesis is also supported by unpublished observations from this laboratory, which demonstrated that neutrophils release a factor or factors that inhibit TNF- production by isolated macrophages (J. M. Daley, unpublished observations). Evidence in the literature provides support for this conclusion. In this connection, Karsunky et al. (10) reported that mice lacking the transcriptional repressor Gfi1 are profoundly neutropenic and respond to endotoxin with greater serum TNF-, IL-1, and IL-10 concentrations than controls. Moreover, similar to findings in cyclophosphamide-induced neutropenia (19), Gfi1 knockout animals had increased mortality vs. controls when injected with LPS (10). Extending the spectrum of proinflammatory responses augmented in neutropenia and demonstrating that the response extends to stimuli other than LPS, Omert et al. (14) reported that hemorrhagic shock in rats made neutropenic with vinblastine resulted in significant increases in hepatic and lung IL-6 and CD-14 mRNA. Thus the proinflammatory bias of neutropenic animals has been shown to be independent of the method used to induce neutropenia and of the stressor imposed on the animal.
The exaggerated TNF- response in neutropenic mice has interesting parallels in human disease. Papadaki et al. (15) reported that uninfected adults with chronic idiopathic neutropenia have increased serum levels of TNF-, IL-1, IL-6, and TGF-1 and that serum cytokine concentrations in these subjects correlate inversely with the number of circulating neutrophils (15). Moreover, neutropenic chemotherapy patients frequently experience febrile episodes in the absence of a demonstrable infectious source (7). It may be that, in addition to their recognized antimicrobial capacity, neutrophils participate in the regulation of innate immunity by downmodulating systemic proinflammatory responses and that some febrile episodes in neutropenic patients may result from an overly exuberant response to noninfectious insults that are not counteracted by a normal neutrophil anti-inflammatory activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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and neutrophils in endotoxin-induced liver injury. Am J Physiol Gastrointest Liver Physiol 265: G1011–G1015, 1993. by proteolytic enzymes released from stimulated human neutrophils. J Immunol 147: 3862–3868, 1991.[Abstract]
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