Departments of 1Medicine, Section of Pulmonary/Critical Care Medicine, and 2Physiology, and 3Alcohol Research Center, Louisiana State University Health Sciences Center, New Orleans
Received 26 June 2002; revised 10 September 2002; electronically published 13 December 2002.
CXC chemokines are major chemoattractants for pulmonary polymorphonuclear leukocyte (PMNL) recruitment. To study the effects of interferon (IFN) on the pulmonary chemokine response to lipopolysaccharide (LPS) challenge, rats were treated with intratracheal IFN- (1 × 105 U/rat) 24 h before an intratracheal LPS (100 g/rat) challenge. Intratracheal LPS caused significant increases in both cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein– 2 in bronchoalveolar lavage (BAL) fluid and pulmonary PMNL recruitment. IFN- enhanced these responses. IFN- also increased LPS-induced tumor necrosis factor (TNF) in BAL fluid. LPS-induced TNF- and CINC mRNA expression in alveolar macrophages was increased by IFN-. CD11b/c and CD18 expression on circulating PMNLs was not affected by IFN-, nor was the chemotaxis of these cells. IFN- increases the pulmonary CXC chemokine response, which may serve as one mechanism underlying enhanced PMNL delivery into the lung.
Presented in part: 98th International Conference of the American Thoracic Society, Atlanta, May 2002 (abstract C105).
The experiments were performed in adherence to National Institutes of Health guidelines on the use of experimental animals. Approval from the Animal Care and Use Committee of the Louisiana State University Health Sciences Center was obtained before initiating these experiments.
Financial support: National Institutes of Health (AA09803); Louisiana State Health Excellence Fund (2000-05-06).
Reprints or correspondence: Dr. Steve Nelson, Dept. of Medicine, Section of Pulmonary and Critical Care Medicine, Louisiana State University Health Sciences Center, 1901 Perdido St., Ste. 3205, New Orleans, LA 70112 .
The effective recruitment of polymorphonuclear leukocytes (PMNL) from the systemic circulation into the alveolar space is a critical component of the pulmonary host defense against bacterial infections [1, 2]. In response to the entry of bacterial pathogens into the terminal airways, alveolar macrophages (AMs) are activated and generate numerous mediators that orchestrate the recruitment of PMNLs from the bloodstream into the alveolar space. These recruited PMNLs provide auxiliary phagocytic defenses to the lung that are critical for the effective eradication of invading pathogens [1, 3]. Clinical observations and experimental studies have consistently shown that impairment of this PMNL response predictably results in an increased susceptibility to pulmonary infection [4 6]. In contrast, enhancement of PMNL delivery into the lung accelerates bacterial clearance, improves resolution of the infection, and increases survival [7 10].
Interferon (IFN) (type II IFN) is a glycoprotein produced by T lymphocytes, natural killer cells, and other cell types [11 13]. This cytokine plays an important role in innate immune defense against bacterial infections [14]. IFN- has been shown to exert broad effects on the functional activities of various immune effector cells, including macrophages and PMNLs [15 17]. We have previously reported that IFN- enhances pulmonary PMNL recruitment in animals with lung infections [3]. However, the mechanisms underlying this effect remain to be defined.
PMNL transendothelial migration is a complex process that involves an intricate interplay of various adhesion molecules expressed by both PMNLs and the endothelium. In addition, the presence of chemoattractants in the infected tissue site is essential for directing PMNL migration. Cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein (MIP)– 2 are CXC chemokines in rats [18]. Studies elsewhere have shown that these 2 chemokines account for the major chemotactic activity for PMNLs in the alveolar space during pulmonary infection and inflammation [19, 20]. IFN- has been shown to prime macrophages for the production of proinflammatory cytokines, such as tumor necrosis factor (TNF) [21, 22]. In vitro studies also have reported that IFN- may alter the chemokine response by macrophages and other types of cells [23]. In the present study, we investigated the effects of local administration of IFN- on the pulmonary CINC and MIP-2 responses to intratracheal lipopolysaccharide (LPS) challenge. We also determined PMNL 2-integrin adhesion molecule expression and chemotaxis in these animals, to further elucidate the mechanism(s) underlying IFN-induced enhancement of pulmonary host-defense function. The results show that IFN- increases both CINC and MIP-2 production in the lung in response to LPS challenge.
MATERIALS AND METHODS
Animals. Male virus antibody– free Sprague Dawley International Gold Standard rats (Charles River Laboratories) with a body weight of 175– 200 g were maintained on a standard laboratory diet and housed in a controlled environment with a 12-h light/dark cycle. Intratracheal IFN- (BioSource International) pretreatment was performed on the rats under ether anesthesia. A midline incision was made above the sternum. The trachea then was exposed by blunt dissection. A 28-gauge needle was inserted into the trachea above the carina, and 0.5 mL of IFN- (10 g in PBS) was instilled. The skin incision then was closed by surgical staples. Control animals were pretreated with an equal volume of intratracheal PBS. Intratracheal LPS (100 g in 0.5 mL of PBS; Difco Laboratories) challenge was given to animals 24 h after the IFN- or PBS pretreatment by the same procedures, as described above. Control rats were intratracheally challenged with an equal volume of PBS without LPS. Depending on the experimental protocol, animals were killed either 90 min or 4 h after the intratracheal LPS or PBS challenge. At the end of each experiment, a heparinized whole blood sample was obtained from the abdominal aorta, and bronchoalveolar lavage (BAL) fluid was obtained. In one group of rats, intratracheal LPS or PBS was administered directly without any pretreatment. The rats were killed 4 h after the challenge, to collect BAL fluid. In another set of experiments, animals were killed 24 h after intratracheal IFN- or PBS administration, to obtain a heparinized whole blood sample. These samples were used for analysis of PMNL adhesion molecule expression and chemotaxis.
BAL and isolation of cells. Lungs were lavaged with a total of 30 mL cold PBS that contained 0.1% dextrose, using 10 mL/lavage. Recovered lavage fluid was centrifuged at 200 g for 5 min. The supernatant of lavaged fluid collected from the first wash (7 mL on average) was aliquoted and stored at -80°C for cytokine and chemokine determinations and analysis of PMNL chemotaxis. The cell pellet from each wash was suspended in PBS that contained 0.1% dextrose and then was combined. The cells were quantified under a light microscope with a hemacytometer. A cell monolayer was prepared by cytocentrifugation, and Wright-Giemsa stain was used to differentiate AMs and pulmonary recruited PMNLs.
Circulating PMNLs were isolated from the whole blood samples using a modification of a procedure described elsewhere [24]. In brief, blood was added to 2% methylcellulose (viscosity of 15 centipoises at 25°C) in PBS at a ratio of 2 : 1 and then was centrifuged at 20 g for 7 min. The white cell– containing supernatant was diluted with 2 v of RPMI 1640 medium without phenol red that contained 2% heat-inactivated FCS (RPMI medium) and centrifuged at 530 g for 10 min. The cell pellet was resuspended in 5 mL of RPMI medium and then layered over 3 mL of Nycoprep 1.077 gradient solution (Accurate Chemical and Scientific). After centrifugation at 530 g for 15 min, the contaminated red cells in PMNL pellet were lysed by Purescript RBC lysing solution (Gentra Systems). PMNLs were washed twice with RPMI medium and then suspended in RPMI medium at a concentration of 5 × 106 cells/mL. The viability of isolated PMNLs was >95%, as assessed by trypan blue exclusion, with a purity >90%, as assessed by morphology using Wright-Giemsa stain.
Measurement of CINC, MIP-2, and TNF-. CINC was determined with a specific ELISA assay, as described elsewhere [25]. MIP-2 and TNF- in plasma were measured in duplicates using the immunoassay kits for rat MIP-2 and TNF- and procedures supplied by the manufacturer (BioSource International).
Sample preparation for real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) assays. Total RNA load from AMs recovered by BAL was extracted with TRIzol reagent (Life Technologies), according to the manufacturer's protocol, with the exception that 1-bromo-3-chloropropane phase separation reagent was substituted for chloroform during the extraction procedure.
Determination of CINC, MIP-2, and TNF- mRNA by real-time quantitative RT-PCR. The primers and probes used for determination of CINC and MIP-2 mRNA were the same as that described elsewhere [26, 27]. Primers and probe used for determination of TNF- mRNA were designed using Primer Express software (PE Applied Biosystems): forward primer, 5-ATG TGC TCC TCA CCC ACA CC-3; reverse primer, 5-TCA TAC CAG GGC TTG AGC TCA-3; and probe, 5-TCA GCC GAT TTG CCA TTT CAT ACC AGG-3. On the basis of the design of the above primers, the length of the amplicon for TNF- was 128 bp.
RNA standards for rat CINC, MIP-2, and TNF- were prepared as described elsewhere [26 28]. Human ribosomal RNA was obtained from PE Applied Biosystems. For each real-time quantitative RT-PCR assay, CINC, MIP-2, TNF-, and ribosomal RNA standard curves (ranging from 103 to 1010 RNA copies per reaction for CINC, MIP-2, and TNF- and ranging from 10-7 to 101 ng RNA/L for ribosomal RNA) were generated.
All reactions were carried out using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). CINC and MIP-2 mRNA were determined by 1-step real-time quantitative RT-PCR protocols, as described elsewhere [26, 27]. TNF- mRNA was measured by 2-step real-time quantitative RT-PCR assay, according to the Taqman Universal PCR Mastermix protocol (PE Applied Biosystems). In brief, reverse transcription was carried out in a total volume of 10 L that contained the following components: 5 L sample RNA (10 ng), 2 L MgCl2 (5 mM), 0.5 L pd(N)6 (25 L/mL), 0.5 L Rnasin (20 U), 0.4 L dNTPs (1 mM), 0.5 L dithiothreitol (5 mM), 1 L 10 times PCR buffer II (1×), and 0.1 L Superscript II RT (20 U). Thermocycling conditions for RT were 10 min at 25°C for equilibration and 30 min at 48°C for RT, followed by heat inactivation for 30 min at 95°C. After RT, 40 L containing the appropriate PCR reagents was added to the original 10 L for RT. The following components were included in the 40-L PCR mix: 25 L Taqman Universal PCR Master Mix (1×), 5 L forward primer (900 nM), 5 L reverse primer (900 nM), and 5 L probe (200 nM). Thermocycling conditions consisted of 2 min at 50°C for optimal buffer equilibration, AmpliTaq Gold DNA polymerase activation for 10 min at 95°C, and 40 cycles of denaturation and annealing/extension (15 s at 95°C followed by 1 min at 60°C). RNA quantities were determined by comparing the cycle threshold of each sample to those of RNA standard curves. CINC, MIP-2, and TNF- mRNA values were normalized to the content of 18s rRNA in each sample and expressed as copies/ng rRNA.
Determination of CD11b/c and CD18 expression. Heparinized whole blood from rats pretreated with or without IFN- was suspended in diluted BAL fluid (1 : 2) from animals challenged with either PBS or LPS for 4 h, and the cell suspension was incubated in vitro at 37°C for 60 min. CD11b/c and CD18 expression on PMNLs were measured by flow cytometry, as reported elsewhere [29]. A FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems) was used for this analysis. Results are expressed as mean channel fluorescence intensity.
Preparation of zymosan-activated serum (ZAS). ZAS was prepared from pooled normal rat serum by adding zymosan (15 mg/mL). The serum mixture was incubated with end-over-end rotation at 37°C for 60 min. After heat inactivation at 56°C for 30 min, the serum mixture was centrifuged (1000 g) at 4°C for 15 min. Aliquots of the supernatant were collected and stored at -80°C.
Determination of PMNL chemotaxis. PMNL chemotaxis was determined with modification [27] of a method reported elsewhere [30]. In brief, isolated PMNLs were suspended in RPMI medium that contained 5 M Calcein AM (Molecular Probes) at a concentration of 5 × 106 cells/mL. The cells were incubated on a cell rocker for 30 min at 37°C. Neutrophils were washed twice with RPMI medium and suspended in PBS that contained 0.1% human serum albumin (PBS-HSA) to a final concentration of 3.0 × 106 cells/mL. BAL fluid and ZAS were diluted at 1 : 10 with PBS-HSA. Samples (30 L) were loaded in triplicate into the wells of the ChemoTx disposable 96-well chemotaxis system (Neuro Probe). A framed filter (containing 3-m diameter pores) was installed. Neutrophil suspension (25 L containing 75,000 cells) was pipetted either onto the top side of the filter directly above each well containing chemotactic solutions or into each empty well directly, and the chamber was incubated for 1 h at 37°C in an atmosphere of 5% CO2. After removing nonmigrated cells from the top side of the filter by gently wiping the filter with wet gauze, the chamber was centrifuged at 400 g for 5 min. Migrated cells or total number of cells in the bottom of the chamber were determined by measuring Calcein fluorescence (excitation, 485 nm; emission, 530 nm) with a FL600 microplate fluorescence reader configured to read from the bottom (Bio-Tek Instruments). The migration of PMNLs is expressed as the percentage of total number of cells loaded.
Statistics. Data are presented as mean ± SE. Sample size is indicated in each figure. Comparisons of data were performed with unpaired Student's t test or 1-way analysis of variance (ANOVA), followed by the Student-Newman Keuls test. If the data did not pass the tests for normality and equal variance, they were log10-transformed to fit ANOVA requirements for normality and homogeneity. Differences were considered to be statistically significant at P < .05.
RESULTS
Pulmonary recruitment of PMNLs. To determine PMNL recruitment into the alveolar space after intratracheal LPS, rats were killed at either 90 min or 4 h after the challenge, to allow for lung lavage to occur. In animals challenged with intratracheal PBS, very few PMNLs were recovered from the BAL fluid . IFN- pretreatment had no effect on the number of PMNLs recovered from the BAL fluid in rats challenged with intratracheal PBS. At 90 min after intratracheal LPS challenge, the total number of PMNLs recovered from the BAL fluid was not altered in rats pretreated with either vehicle or IFN-. At 4 h after intratracheal LPS challenge, the total number of cells recovered by BAL was significantly increased. This increase in total cell number was primarily caused by an increase in the number of PMNLs. PMNLs constituted >75% of total cells recovered from the BAL fluid in these animals. IFN- pretreatment caused a significant enhancement (>3-fold) of PMNL recruitment into the alveolar space at 4 h after intratracheal LPS challenge, and 94% of the cells recovered from the BAL fluid were PMNLs.
fig.ommitted
Table 1. Cells (×106) recovered by bronchoalveolar lavage.
CINC, MIP-2, and TNF-α response in BAL fluid. CINC and MIP-2 are potent CXC chemokines in the rat lung for PMNLs. In control rats challenged with intratracheal PBS, CINC concentration in BAL fluid was very low irrespective of IFN- pretreatment (<500 pg/mL, data not shown). Intratracheal LPS caused a significant increase in CINC concentration in BAL fluid at 90 min and 4 h after the challenge . IFN- pretreatment significantly enhanced the CINC response to LPS challenge in the lung at both 90 min (1.9-fold) and 4 h (3.4-fold). Similarly, MIP-2 concentration in BAL fluid was low (<600 pg/mL, data not shown) in control rats challenged with intratracheal PBS. IFN- pretreatment had no effect on this baseline MIP-2 production. Intratracheal LPS challenge resulted in a marked increase in MIP-2 concentration in BAL fluid at 90 min and 4 h after challenge. IFN- pretreatment did not enhance the LPS-induced MIP-2 response in the lung at 90 min after challenge. However, intratracheal LPSinduced increase in MIP-2 concentration in BAL fluid was significantly enhanced (2.1-fold) in animals pretreated with IFN- at 4 h after the challenge. We also determined the TNF- response in the lungs of these animals. Only trace amounts of TNF- (<150 pg/mL, data not shown) were detected in the BAL fluid of intratracheal PBS challenged rats that received either vehicle or IFN- pretreatment. Intratracheal LPS induced a significant increase in TNF- production in the lung at 90 min after the challenge. Four hours after intratracheal LPS challenge, the TNF- concentration in the BAL fluid was slightly lower than that at 90 min but remained higher, compared with values in PBS-challenged control rats. IFN- pretreatment significantly enhanced pulmonary TNF- production at 90 min (2.9-fold) and 4 h (6.2-fold) after intratracheal LPS challenge.
fig.ommitted
Figure 1. Effects of interferon (IFN) pretreatment on the pulmonary cytokine-induced neutrophil chemoattractant (CINC), macrophage inflammatory protein (MIP)2, and TNF- responses to intratracheal lipopolysaccharide (LPS) challenge. Values are mean ± SE; n = 5 for each group. PBS/LPS, pretreated with intratracheal PBS followed by intratracheal LPS challenge; IFN/LPS, pretreated with intratracheal IFN- followed by intratracheal LPS challenge. Data were analyzed with 1-way analysis of variance followed by the StudentNewman Keuls test.
CINC, MIP-2, and TNF- response in plasma. The effects of IFN- on plasma CINC, MIP-2, and TNF- response to intrapulmonary LPS challenge also were determined. Similar to our previous observations [31], plasma concentration of CINC was low (518 ± 127 pg/mL) in control rats challenged with intratracheal PBS. Intrapulmonary challenge with LPS caused a significant increase in CINC in the plasma at both 90 min and 4 h after challenge . IFN- did not affect plasma CINC concentration in PBS-challenged control rats (data not shown) but significantly enhanced the increases in plasma CINC in intratracheal LPS-challenged animals at both 90 min (2.6-fold) and 4 h (2.9-fold). Plasma MIP-2 was not detectable in any of these studies. A trace amount of TNF- was detected in the plasma of rats that was not altered by either IFN- pretreatment or intratracheal LPS challenge (data not shown).
fig.ommitted
Figure 2. Effects of interferon (IFN) pretreatment on the plasma cytokine-induced neutrophil chemoattractant (CINC) response to intratracheal lipopolysaccharide (LPS) challenge. Values are mean ± SE; n = 5 in each group. PBS/LPS, pretreated with intratracheal PBS followed by intratracheal LPS challenge; IFN/LPS, pretreated with intratracheal IFN- followed by intratracheal LPS challenge. Data were analyzed with 1-way analysis of variance followed by the Student-Newman Keuls test.
CINC, MIP-2, and TNF-α mRNA expression by AMs. In addition to determining the protein concentration of CINC, MIP-2, and TNF- in the BAL fluid, we also measured alveolar macrophage messenger RNA expression of these inflammatory mediators using real-time RT-PCR. The baseline expression of CINC, MIP-2, and TNF- mRNA (4.0 × 103 ± 3.6 × 103, 93.2 × 103 ± 65.8 × 103, and 7.6 × 103 ± 6.2 × 103 copies/ng rRNA, respectively) by AMs was very low. IFN- pretreatment did not cause an up-regulation of cell mRNA expression for these 3 cytokines (data not shown). CINC mRNA expression by AMs 90 min after intratracheal LPS challenge was 25-fold higher than the control value of PBS-challenged animals . IFN- significantly enhanced intratracheal LPS-induced up-regulation of CINC mRNA expression by AMs. Intratracheal LPS caused a marked up-regulation of MIP-2 mRNA expression by AMs (35.7-fold) at 90 min after challenge. This LPS-induced up-regulation of MIP-2 mRNA expression by AMs was higher in IFN-pretreated rats but did not reach statistical significance. Intratracheal LPS also caused a significant up-regulation of TNF- mRNA expression by AMs (66.7-fold) at 90 min after the challenge. IFN- pretreatment significantly enhanced LPS-induced up-regulation of TNF- mRNA expression by AMs (3-fold) 90 min after challenge.
fig.ommitted
Figure 3. Effects of interferon (IFN) pretreatment on cytokine-induced neutrophil chemoattractant (CINC), macrophage inflammatory protein (MIP)2, and tumor necrosis factor (TNF) mRNA expression in alveolar macrophages at 90 min after intratracheal lipopolysaccharide (LPS) challenge. Values are mean ± SE; n = 5 for each group. IFN/LPS, pretreated with intratracheal IFN- followed by intratracheal LPS challenge; PBS/LPS, pretreated with intratracheal PBS followed by intratracheal LPS challenge. Data were analyzed with 1-way analysis of variance followed by the Student-Newman Keuls test.
PMNL 2-integrin adhesion-molecule expression and chemotaxis. To determine whether there was an effect of IFN- on PMNL function, we measured 2-integrin adhesion-molecule expression in PMNLs from rats treated with or without IFN-. As shown in , exposure to BAL fluid of LPS-challenged rats caused a marked up-regulation of both CD11b/c and CD18 expression on PMNLs in comparison to those of PMNLs exposed to the BAL fluid of PBS-challenged animals. In vivo treatment with IFN had no effect on either CD11b/c or CD18 expression on PMNLs after incubation of these cells with BAL fluid collected from either PBS- or LPS-challenged rats. The effect of IFN- treatment on PMNL chemotaxis was also tested. ZAS-induced chemotaxis was similar for PMNLs obtained from PBS- and IFN-treated animals . Of interest, chemotaxis toward the BAL fluid of intratracheal LPS-challenged rats was lower for PMNLs obtained from IFN-treated rats compared with the PMNLs from vehicle-treated control animals.
fig.ommitted
Figure 4. Polymorphonuclear leukocytes (PMNL) 2-integrin adhesion molecule expression. Values are mean ± SE; n = 4 for each group. IFN/LPS, PMNLs from interferon-treated rats incubated with the bronchoalveolar lavage (BAL) fluid of rats challenged with intratracheal lipopolysaccharides; IFN/PBS, PMNLs from IFN-– treated rats incubated with the BAL fluid of rats challenged with intratracheal PBS; PBS/LPS, PMNLs from PBS-treated rats incubated with the BAL fluid of rats challenged with intratracheal LPS; PBS/PBS, PMNLs from PBS-treated rats incubated with the BAL fluid of rats challenged with intratracheal PBS. Data were analyzed with 1-way analysis of variance followed by the StudentNewman Keuls test. Columns with different letters are statistically different (P < .05).
fig.ommitted
Figure 5. Polymorphonuclear leukocyte (PMNL) chemotaxis. Values are mean ± SE; n = 4 in each group. PBS, PMNLs from rats treated with intratracheal PBS; IFN, PMNLs from rats treated with intratracheal interferon-; BALF, bronchoalveolar lavage (BAL) fluid of rats challenged with intratracheal LPS for 4 h; ZAS, Zymosan-activated serum. Data were analyzed with unpaired Student's t test.
DISCUSSION
Consistent with our previous studies, pretreatment with IFN- resulted in a significantly increased influx of PMNLs into the LPS-challenged lung. At 4 h after an intratracheal LPS challenge, large numbers of PMNLs were recovered from the BAL fluid of rats. Although IFN- did not directly evoke an inflammatory response in the lung, it did markedly enhance PMNL recruitment into the alveolar space in response to intratracheal LPS. Elsewhere, we have shown that pretreatment of rats with intratracheal IFN- 24 h before aerosol challenge with Pseudomonas aeruginosa significantly enhances pulmonary recruitment of PMNLs. Pulmonary clearance of P. aeruginosa is also accelerated in IFN-pretreated animals [3]. In another set of studies, we have shown that intratracheal administration of a recombinant adenoviral vector encoding murine IFN- complementary DNA results in prolonged expression of biologically active IFN- in rat lung. These animals recruit significantly more PMNLs into the alveolar space after an intrapulmonary challenge with bacteria. Bacterial killing activity in the lung also is enhanced in these animals [32, 33]. Although these studies showed that IFN- enhances PMNL influx into the lung during pulmonary infection and inflammation, the underlying mechanisms remained to be defined.
PMNL migration into tissue sites from the systemic circulation is a complex process that requires the presence of chemoattractants. An established concentration gradient of chemoattractants from the tissue site to the bloodstream is necessary for initiating PMNL transendothelial migration. CINC and MIP-2 are potent CXC chemokines [18, 34] that account for the major chemotactic activity for PMNLs in rat lung during pulmonary infection and inflammation [19, 20]. In the present study, we investigated the effects of IFN- on the pulmonary CINC and MIP-2 responses to intratracheal LPS challenge. The results show that intrapulmonary administration of recombinant rat IFN- 24 h before intratracheal LPS challenge significantly enhances both CINC and MIP-2 production in the alveolar space of the rats. These data suggest that the increase in CXC chemokine production in the lung may play an important role in IFN-induced enhancement of pulmonary PMNL recruitment after intratracheal LPS challenge.
AMs are resident phagocytes in the alveolar space that produce CINC and MIP-2 in response to LPS [25]. In the present study, we determined the effects of IFN- pretreatment on CINC and MIP-2 mRNA expression by AMs after intratracheal LPS challenge. AMs obtained from IFN-pretreated rats at 90 min after intratracheal LPS challenge showed an enhanced up-regulation of CINC mRNA expression, compared with those of LPS-challenged rats in the absence of IFN- pretreatment. MIP-2 mRNA expression by AMs obtained from IFN- pretreated rats at 90 min after intratracheal LPS challenge trended higher, but the difference did not reach statistical significance. These results suggest that IFN- modulates AM CXC chemokine response at the level of gene expression.
The mechanism(s) by which IFN- modulates AM CXC chemokine gene expression remain to be elucidated. TNF- is an early response cytokine and a potent stimulator for both CINC and MIP-2 production by various types of cells [35 37]. In our present study, TNF- concentration in the BAL fluid of rats challenged with intratracheal LPS was significantly increased. IFN- pretreatment markedly enhanced the pulmonary TNF- response in animals challenged with intratracheal LPS. IFN- pretreatment also markedly enhanced the up-regulation of TNF- mRNA expression by the AMs. These data suggest that the enhanced CXC chemokine response to LPS challenge in the lung of rats pretreated with IFN- may be mediated (at least in part) by the increase in TNF- in the alveolar space of these animals. In support of this hypothesis, our previous studies have shown that neutralizing TNF- in the alveolar space by antiTNF- antibody blocks IFN-induced enhancement of pulmonary PMNL recruitment in animals challenged with intrapulmonary bacteria [3].
At the present time, reported observations about the direct effects of IFN- on macrophage CXC chemokine responses are inconsistent. Incubation of peritoneal macrophages from LPS-responsive C3H/OuJ mice with IFN- has been reported to inhibit MIP-2 and KC responses by these cells to LPS stimulation [23]. In contrast, other investigations have shown that exposure of alveolar and peritoneal macrophages from LPS-hyporesponsive C3H/HeJ mice to IFN- in vitro enhances MIP-2 responses to LPS stimulation by both types of macrophages [38].
Binding of IFN- to its receptor on cell surface is an initial step in triggering cell biological responses [39]. The engagement of IFN- to its receptor causes receptor dimerization, phosphorylation, and internalization [39, 40]. Recently, evidence suggests that multiple signal transduction pathways including Janus activated-signal transducer and activator transcription factor, mitogen-activated protein kinase, and protein kinase C signaling systems may be involved in mediating IFN-induced cell functional alteration and gene induction in various types of cells [17, 40 42]. At present, however, information about IFN-induced activation of intracellular signaling systems leading to modulation of CINC and MIP-2 production by AMs and other types of pulmonary cells is limited. Further study on the involved signaling cascades will improve our understanding of the mechanisms by which IFN- regulates pulmonary chemokine responses during bacterial infections.
An important feature of the pulmonary host defense response is the differential compartmentalization of certain cytokines and chemokines [1]. Our previous studies have shown that TNF- and MIP-2 are confined within the lung in animals challenged with either intratracheal LPS or bacteria, whereas CINC produced in the alveolar space can enter the bloodstream [26, 31]. In the present study, plasma TNF-α and MIP-2 were not elevated in rats challenged with intratracheal LPS. Plasma CINC concentration was increased significantly after intratracheal LPS challenge and was significantly enhanced by IFN- pretreatment. These data further support our previous observations.
Alteration of PMNL sensitivity to chemotactic stimuli also plays an important role in the modulation of tissue PMNL recruitment [43]. 2-integrin adhesion molecules CD11b/CD18 expressed by PMNLs mediate firm attachment of PMNLs to the endothelium and the subsequent transendothelial migration of PMNLs. Studies have shown that IFN- modulates several PMNL activities, including bacterial killing, generation of reactive oxygen species, expression of surface receptors, and cellular calcium influx [17, 44 47]. We determined the effects of IFN- treatment on PMNL CD11b/c and CD18 expression in response to the BAL fluid of rats challenged with intratracheal LPS. PMNL chemotaxis to chemotactic stimuli also was determined in animals treated with or without IFN-. The results show that IFN- treatment did not affect the up-regulation of CD11b/c and CD18 expression on circulating PMNLs in response to the BAL fluid of rats challenged with intratracheal LPS. Of interest, circulating PMNLs from IFN-treated rats exhibited decreased chemotaxis toward the BAL fluid of intratracheal LPS-challenged animals. The mechanisms underlying this inhibition of PMNL chemotaxis in IFN-treated rats remain unclear. One possible explanation is that selected PMNLs in IFN-treated rats may become activated and adhere to the endothelium of the blood vasculature. The PMNLs remaining in the bloodstream could then represent a subpopulation of hyporeactive PMNLs.
In summary, IFN- is an important immunomodulator that enhances the innate immune response of the lung. Local administration of IFN- enhances CXC chemokine production in the alveolar space in response to LPS challenge. The modulation of AM CXC chemokine response involves the up-regulation of chemokine gene expression. IFN-enhanced CXC chemokine production in the alveolar space may serve as one mechanism underlying the augmentation of pulmonary PMNL recruitment during lung infection. In humans, aerosol delivery of IFN- has been shown to activate AMs in a compartmentalized fashion [48]. IFN- has also been reported to be useful for the prophylaxis and treatment of secondary infections in immunocompromised hosts, including individuals with AIDS, trauma, and chronic granulomatous disease [49]. These data suggest that local administration of IFN- may be a useful adjuvant therapy in patients by amplifying the proinflammatory response of the lung to invading pathogens.
Acknowledgments
We thank Amy B. Weinberg and Rhonda R. Martinez for their excellent technical assistance. We also thank Howard L. Blakesley for his help in data analysis. We are grateful to Connie P. Porretta for her expert assistance in flow cytometry.
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(Ping Zhang Lee J. Quinton Gregory J. Bagby Warren R. Summer and Steve Nelson)
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