Elevated serum and bronchoalveolar lavage ... - Wiley Online Library

British Journal of Haematology, 2000, 111, 482±490

Elevated serum and bronchoalveolar lavage fluid levels of interleukin 8 and granulocyte colony-stimulating factor associated with the acute chest syndrome in patients with sickle cell disease Miguel R. Abboud, 1 Ellen C. Taylor, 1 David Habib, 1 Terrie Dantzler-Johnson, 1 Sherron M. Jackson, 1 Fushen Xu, 1 Joseph Laver, 1 and Samir K. Ballas 2 1 Division of Pediatric Hematology/Oncology at the Medical University of South Carolina, Charleston, SC, and 2 The Cardeza Foundation for Hematologic Research, Jefferson Medical College, Philadelphia, PA, USA Received 23 February 2000; accepted for publication 21 June 2000

Summary. The role of cytokines in the development of acute chest syndrome (ACS) in patients with sickle cell disease (SCD) was studied. Serum interleukin 8 (IL-8) levels were elevated in 14 episodes and undetectable in six out of 20 episodes of ACS in 19 patients with SCD. In contrast, IL-8 levels were undetectable in the sera of 29 control patients with SCD studied during routine clinic visits or hospitalization for vaso-occlusive crises. The differences in mean IL-8 levels and the proportion of patients with detectable levels between the two groups were highly significant (P , 0´0001 and 0´04 respectively). The mean IL-8 level in bronchial fluid samples from children with ACS was also significantly higher than that in sickle cell patients

undergoing elective surgery (5500 ^ 1400 pg/ml vs. 1900 ^ 470 pg/ml, P 0´03). Granulocyte colony-stimulating factor (G-CSF) (2000 ^ 1700 pg/ml) was present in five out of six samples of bronchial fluid, but not serum, from children with ACS. All but one of the patients with ACS studied were negative for the Duffy red cell antigen, which is a receptor that binds and inactivates IL-8 and other chemokines. These findings suggest that IL-8 and G-CSF may play a role in the development of the ACS and the complications associated with it.

Acute chest syndrome (ACS) is characterized by fever, leucocytosis, respiratory symptoms and a new infiltrate on chest radiograph (CXR). It is the most common cause of death and the second most common cause of hospitalization in patients with sickle cell disease (SCD) (Sprinkle et al, 1986; Castro et al, 1994; Vichinsky & Styles, 1996; Vichinsky et al, 1997). A majority of SCD patients will experience at least one episode of ACS and repeated episodes can lead to chronic lung damage (Powars et al, 1988). It is known that patients with acute chest syndrome respond rapidly to blood transfusion (Mallouh & Asha, 1988). The rapidity of this response has led some to postulate that this effect is as a result of the removal of circulating cytokines, which cause lung injury, by the transfused red cells.

Cytokines, particularly interleukin 8 (IL-8) and granulocyte colony-stimulating factor (G-CSF), are involved in the pathogenesis of neutrophil-mediated lung injury. IL-8 is a potent mediator of neutrophil chemotaxis. It also induces the release of storage enzymes from granules and increases the production of reactive O2 metabolites via the respiratory burst (Baggiolini et al, 1994). G-CSF significantly modulates the number and function of neutrophils (Dale et al, 1995). Elevated levels of IL-8 have been found in the bronchoalveolar lavage fluid (BALF) and serum of patients with adult respiratory distress syndrome (Chollet-Martin et al, 1996), community-acquired pneumonia (Bohnet et al, 1997), asthma (Nocker et al, 1996) and sarcoidosis (Takizawa et al, 1997). In addition, lung reperfusion injury in rabbits was prevented by treatment with a monoclonal antibody against IL-8 (Sekido et al, 1993). High BALF levels of IL-8 and G-CSF have also been reported in human immunodeficiency virus (HIV)-infected persons with bacterial or pneumocystis pneumonia (GruÈnewald et al, 1993;

Correspondence: Miguel R. Abboud, Department of Pediatrics, Division of Hematology/Oncology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-3311, USA. E-mail: [email protected]

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Keywords: interleukin 8, G-CSF, acute chest syndrome, cytokines.

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IL-8 and G-CSF Levels in Patients with SCD and ACS

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Jefferson University were the subjects of this study. Blood samples from patients and controls were obtained at the time of illness or during clinic visits when blood samples were drawn as required for clinical care. No extra venepuntures were performed for this study and, thus, it was deemed to be exempt from Institutional Review Board (IRB) approval. BALF samples were obtained by bronchoscopy, after informed consent was obtained, on patients enrolled on the National Acute Chest Study. This study was approved by the IRB at MUSC. Patients with ACS. Serum and/or BALF samples were obtained during 23 episodes of ACS from 17 children admitted to MUSC from February 1995 to October 1996 and from five adults admitted to the Thomas Jefferson Hospital from September 1994 to August 1997. An additional patient, a girl with very severe ACS (patient 22), was studied in mid-1998 at MUSC. Patient characteristics are summarized in Table I. Twenty patients had HbSS disease, one patient had HbSC disease and one patient had HbSb0 thalassaemia. There were 11 females and 11 males and their mean age was 16´5 ^ 1´5 years. One patient was receiving chronic transfusion treatment at the time of ACS. Serum samples were obtained again 1±6 months post ACS in 10 patients (while in a healthy state) who had elevated serum IL-8 levels at the time of diagnosis. One adult patient had serial IL-8 levels measured. Ten BALF samples were

Lipschik et al, 1993; Benfield et al, 1995; Krarup et al, 1997). The Duffy blood group antigen has been recognized to be a receptor that inactivates bound IL-8 and other chemokines (Darbonne et al, 1991; Horuk et al, 1993). The Duffy blood group has two major antigens, Fya and Fyb (Hadley & Peiper, 1997). While the vast majority of Caucasians are Duffy blood group positive, this antigen is lacking [Fy (a2b2)] on the red blood cells of most African-Americans (Horuk et al, 1993; Hadley & Peiper, 1997) and patients with SCD (Vichinsky et al, 1990). Thus, it is conceivable that, in the development of ACS, an initial insult to the lung leads to the release of inflammatory cytokines, including IL-8. As most of the patients lack the Duffy antigen, the action of the cytokines will be unopposed, leading to the severe picture observed in the acute chest syndrome. This study was conducted to determine whether IL-8 and G-CSF play a role in the development of acute chest syndrome in adults and children with sickle cell disease. Serum and BALF levels of both cytokines were found to be elevated in patients with ACS when compared with control groups. PATIENTS AND METHODS Children and adults with sickle cell disease seen at the Medical University of South Carolina (MUSC) and Thomas Table I. Characteristics of patients with acute chest syndrome.

Patient 9 11 12 10 2 22 4* 3 13 15 16 14 6 5 7 18 1* 8 21 19 20 17

Age/ sex

Hb (g/dl)

WBC ( 109/l)

Serum IL-8

5/Male 5/Male 5/Female 6/Male 11/Male 11/Female 13/Female 14/Male 15/Female 15/Female 15/Male 16/Male 16/Female 16/Female 19/Female 19/Female 20/Male 20/Male 21/Male 24/Female 26/Male 27/Male 34/Female

8´6 5´7 8´4 8´1 6´7 8´7 10´8 8´7 8´1 5´7 3´0 9´4 7´7 8´4 6´8 6´0 8´2 11´9 7´9 7´7 8´2 7´5 6´5

21´7 46´4 45´9 30´8 12´2 59´4 50´0 23´8 25´7 15´4 55´0 21´4 26´5 7´5 35´0 20´4 20´5 18´1 31´8 10´6 17´1 12´4 24´1

15 467 Undetectable 900 2200 Undetectable 100 9517 Undetectable Undetectable ± ± ± Undetectable 77 467 304 235 8300 Undetectable 14 26 72 13 15 868

BALF IL-8

±

867 6626

600 ± 180 000 ± 6334 ± 7816 134 000 6130 8267 Undetectable ± ± ± ± ± ± ± ± ±

G-CSF ± ± ± ± ± ± ± ±

390 307

71 1206 10 341 Undetectable ± ± ± ± ± ± ± ± ±

Lobes

Pleural effusion

Trans.

Duffy

2 2 1 2 1 4 3 2 2 4 2 2 2 3 1 1 1 2 3 2 1 2 2

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No No Yes No Yes No No No

Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes No Yes Yes No No Yes No No No

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive

*Patient 1 Hb Sb0; patient 4 HbSC; all others HbSS. Concentrations of IL-8 and G-CSF in pg/ml. Lobes, number of lobes involved; Trans, if patients were transfused for episode. Aetiology: patient 3, pulmonary fat embolus; patient 4, Mycoplasma; patient 9, Chlamydia; patient 15, parvo B19. q 2000 Blackwell Science Ltd, British Journal of Haematology 111: 482±490

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obtained, by bronchoscopy from nine children and through an endotracheal tube in one patient, during episodes of ACS. Seven of these patients had concomitant serum samples available. Cells were obtained from this fluid and the supernatant frozen at 2708C until the time of cytokine analyses. Control group. Thirty serum samples were collected from 29 patients with SCD, one of whom had HbSC, while the others had HbSS. Their characteristics are summarized in Table II. There were 16 female and 13 male patients and the mean age for this group was 24´9 ^ 2´2 years. This was significantly higher than the mean age of patients with ACS (P 0´008) as there were more adults in the control group and more children in the ACS group. This may be because ACS is more common in younger patients, while fewer children were admitted for painful crises during the study period. Thirteen samples were obtained during routine clinic visits and four samples prior to regular scheduled transfusions for the long-term management of strokes. The remaining 13 samples were obtained while patients were hospitalized for vaso-occlusive crises (VOC). One patient

contributed two samples, one when an inpatient for VOC and the other in clinic. Transfused and non-transfused patients were studied to determine if there were any differences in cytokine levels that could be attributed to treatment with transfusions. No BALF was obtained from asymptomatic adults or children with SCD in view of the invasive nature of the procedure. Patients undergoing surgery. Eleven patients with HbSS who underwent surgery under general anaesthesia were also studied after informed consent was given. These patients were to serve as controls of bronchial fluid cytokine levels as the lavage could be easily performed in patients who were otherwise well. This was carried out with the recognition that ACS is often a complication of general anaesthesia and high levels of cyokines may be related to the development of ACS. The patients were seven boys and four girls with a mean age of 11´5 ^ 1´5 years. Seven patients had laparoscopic cholecystectomies and each of the remaining four patients had splenectomy, G-tube placement, central line placement and endoscopy. Eight patients received simple transfusions to increase their haemoglobin

Table II. Characteristics of control patients. Patient

Age/sex

Hb (g/dl)

WBC ( 109/l)

Serum IL-8

Chron. Trans.

Previous ACS

In Hosp. VOC

Duffy

1 3* 6 5 2 8 10 4 7 9 20 13 21 22 14 24 16 12 23 26 19 11 15

7/Female 7/Female 9/Female 13/Male 14/Male 14/male 14/female 15/Female 17/Male 17/ Male 17/Male 20/Female 21/Male 22/Male 22/Female 24/Female 26/Male 30/Female 30/Male 31/Male 32/Male 34/Female 34/Female 34/Female 34/Female 35/Male 36/Female 37/Female 42/Female 65/Female

9´4 9´8 7´0 7´6 8´7 9´3 7´0 8´9 7´8 ± 6´1 ± 7´3 6´8 9´0 10´2 9´0 7´9 7´1 7´9 8´8 7´4 8´6 7´0 6´0 8´9 6´9 8´7 6´6 8´2

9´7 12´7 9´8 7´6 15´3 11´1 13´6 14´6 16´4 ± 10´7 ± 9´9 34´3 14´7 19´8 15´5 9´7 14´0 12´2 15´4 10´1 12´4 9´7 15´4 12´9 16´0 10´9 8´6 7´5

Undetectable Undetectable Undetectable Undetectable Undetectable undetectable undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable

Yes No No No Yes No No Yes Yes No No No No No No No No No No No No No No No No No No No No No

Yes Yes No No Yes No Yes Yes Yes Yes Yes No Yes Yes No No Yes No No Yes No Yes No No Yes Yes No Yes No No

No No No No No No No No No No Yes Yes Yes Yes No No No Yes Yes Yes Yes Yes No Yes Yes Yes No No Yes No

± Negative Negative Positive ± Negative Negative ± ± Negative Negative Positive Negative Negative Negative Positive Negative Negative Negative Negative Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative

29 28 18 17 27 25

*Patient 3 HbSC; all others HbSS. Chron Trans, indicates patients on transfusions for CVA; Prev. ACS, indicates whether patients had ACS in the 3 years prior to the study; In hosp. VOC, indicates whether patients were hospitalized for VOC at the time of study. q 2000 Blackwell Science Ltd, British Journal of Haematology 111: 482±490

IL-8 and G-CSF Levels in Patients with SCD and ACS (Hb) to 10 g/dl and three patients were on chronic transfusion protocols because of strokes or sequestration before surgery. Blood was obtained before surgery, intraoperatively (30±60 min after intubation) and 12±24 h after surgery. The endotracheal tube was lavaged with 15±50 ml of normal saline and the lavage fluid collected. Patients with pneumonia. Four children who did not have sickle cell disease, but who were admitted because of severe pneumonia and hypoxia, were studied. The patients were three boys and one girl and their mean age was 10´6 ^ 1´3 years. Blood samples (eight in total) were obtained at different times during their hospitalization when symptoms were severe. One patient had a high cold agglutinin titre and the cause of pneumonia was unknown in three patients. Blood cultures were negative in all four patients. Quantification of cytokines. All samples of serum or BALF were frozen at 2708C and thawed only once at the time of the assay. All serum samples were assayed in duplicate. IL-8, tumour necrosis factor a (TNF-a), IL-1b, G-CSF and granulocyte±macrophage (GM)-CSF levels in serum, BALF and alveolar macrophage supernatant were determined using sensitive enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Minneapolis, MN, USA). The assay sensitivities were 10 pg/ml, 0´18 pg/ml, 0´5 pg/ml, 0´8 pg/ml and 0´36 pg/ml for the above cytokines respectively. Briefly, 100±200 ml samples were dispensed into 96-well microtitre plates of the `Quantokine' kit and incubated at room temperature for 2 h. The plates were then rinsed four times with wash buffer and incubated for 2 h with 200 ml of anti-cytokine±horseradish peroxidase conjugate that corresponded to each cytokine tested. The bound enzyme was then detected by incubation with tetramethylbenzidine and hydrogen peroxide as substrate and quantified by using Microplate Reader (Bio-Rad, Melville, NY, USA). Alveolar macrophages recovery and culture. The pooled lavage fluid was centrifuged and the cell pellet was resuspended in serum-free Roswell Park Memorial Institute (RPMI)-1640 culture medium and plated on 100-mm plastic tissue culture dishes for 15 min at 378C in a humidified 5% CO2 incubator. Non-adherent cells were then removed and the adherent cells were washed twice with 10 ml of cold sterile phosphate-buffered saline (PBS). The purity of the macrophages were greater than 98%, the viability was determined by trypan blue. Isolation of total cellular RNA. Total cellular RNA was extracted by using a RNA purification kit (Qiagen, Chatsworth, CA, USA). Briefly, samples were first lysed and homogenized under highly denaturing conditions that inactivated RNAases to ensure isolation of intact RNA. After adjustment of buffer conditions to promote adsorption, the sample was applied to an RNeasy spin column. Total RNA adsorbed, while contaminants were efficiently washed away. High-quality RNA was then eluted in 30 ml or more of water. The RNeasy procedure provides an enrichment for mRNA as small RNAs such as tRNA and 5S RNA were not isolated. Reverse transcription of RNA. Levels of IL-8 expression were

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determined by reverse transcription of RNA and quantitative polymerase chain reaction (PCR) was used for determination of relative gene expression. The method was previously described (Ponte et al, 1984). Each sample contained 1 mg of total cellular RNA, 50 mmol/l Tris/HCl pH 8´3, 75 mmol/l KCl, 10 mmol/l MgCl2, 0´5 mmol/l spermidine, 10 mmol/l dithiothreitol, 1 mmol/l of each dNTP (dATP, dTTP, dGTP and dCTP), 20 U of placental ribonuclease inhibitor (RNAsin), 1 mg of (dT) 15±18 primer and 25 U of avian myeloblastosis virus reverse transcriptase in a final volume of 12´5 ml. After incubation at 428C for 60 min, the samples were heated for 5 min at 948C to terminate the reactions and stored at 2208C until used. Quantitative PCR for the determination of relative IL-8 gene expression. Oligonucleotide primers were constructed from the cDNA sequences of IL-8 and b-actin cDNA (Ponte et al, 1984; Matsushima et al, 1988). The sequences of the IL-8 primers were: (1) 5 0 -ATT TCT GCA GCT CTG TCT GAA-3 0 (sense), corresponding to bases 144±164 of the cloned fulllength sequence, and (2) 5 0 -TGA ATT CTC AGC CCT CTT CAA-3 0 (antisense), which anneals to bases 378±398. The b-actin primers were: (1) 5 0 -TCC TGT GGC ATC CAC GAA ACT-3 0 (sense), spanning bases 852±872, and (2) 5 0 -GAA GCA TTT GCG GTG GAC GAT-3 0 (antisense), spanning bases 1146±1166. Each sample contained the upstream and downstream primers (0´7 mmol/l of each primer). Statistical analysis. Means of different groups were compared using paired or unpaired t-test whenever appropriate. Proportions were compared using Fisher's exact two-tailed test. Pearsons correlation coefficients were used to describe linear relationships between serum IL-8 levels and the lowest PaO2. RESULTS Clinical picture of ACS Nine of the 16 children studied were admitted with ACS, the remaining seven were admitted for painful crises, while all five adults were admitted for VOC. During the episodes of ACS the following symptoms were seen: 12 children (75%) had a cough, four children (30%) had shortness of breath, 11 (69%) children had chest pain and nine children (56%) had fever. All five adults developed cough, shortness of breath and chest pain; two had low-grade fever. The most severely affected child (patient 22) developed acute chest syndrome 48 h after hospital admission for pain. This patient developed respiratory distress and progressed to failure requiring mechanical ventilation. She responded to exchange tranfusion and was extubated 72 h later. The clinical course and laboratory values of these patients are summarized in Table I. Haemoglobin values on diagnosis ranged from 3 g/dl to 11´9 g/dl with a mean of 7´95 ^ 0´4 g/dl. Fifteen of the 22 patients received simple transfusions and one underwent exchange transfusion. Haemoglobin levels on discharge ranged from 5´2 g/dl to 11´4 g/dl with a mean of 9´0 ^ 0´3 g/dl. The haemoglobin levels on discharge were significantly higher than at the time of onset of ACS (P 0´004). The white blood cell (WBC) counts were elevated at diagnosis and ranged from

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7´5 109/l to 59´4 109/l with a mean of 27´5 ^ 3´0 109/l. On discharge they were significantly decreased and ranged from 10´0 109/l to 27´6 109/l with a mean of 15´4 ^ 1´6 (P 0´002). Platelet counts at diagnosis ranged from 197 109/l to 1335 109/l with a mean of 475 ^ 64. On discharge they ranged from 214 109/l to 1150 109/l with a mean of 595 ^ 60 (P 0´057). Eighteen patients had arterial blood gases obtained at diagnosis. Their PaO2 ranged from 42 mmHg to 98 mmHg with a mean of 71 ^ 5 mmHg. Eighteen patients had O2 saturations at diagnosis ranging from 74% to 99% with a mean of 89 ^ 1%. On discharge they were significantly higher and ranged from 96% to 100% with a mean of 97´5 ^ 0´4% (P , 0´001). Lobe involvement was as follows: one child had one lobe involved, nine children (56%) had two lobes involved, four children (25%) had three lobes involved and two children had four lobes involved. Twelve children had pleural effusions. One child (patient 22) had complete opacification of the right lung and left lower lobe. There were six episodes of ACS among five adult patients studied. Two patients had a single lobe involved, one of these patients (during a second episode) developed bibasilar involvement, one patient had bibasilar involvement, one patient had bibasilar involvement with bilateral pleural effusions and one patient developed complete opacification of the right lung and left lower lobe consolidation.

Other cytokines Serum levels of IL-1b and TNF-a were measured in five patients with ACS and 10 control children with SCD and were not significantly different between the two groups. Serum levels of G-CSF and GM-CSF were undetectable in 17 controls with SCD and eight patients with ACS.

Aetiology of ACS An extensive work-up to determine the aetiology of ACS including blood and urine cultures, Mycoplasma, parvovius B-19 and Chlamydia tests, urine fat analysis and a search for lipid-laden macrophages was negative in 12 out of 16 children studied. Four children had the following aetiologies: fat embolism in one, parvovirus B-19, Mycoplasma and Chlamydia infection in one each of the remaining children. No aetiologies could be determined for the five adult patients.

IL-8 and G-CSF in broncholaveolar lavage IL-8 was found to be elevated in 9 out of 10 samples of BALF from children with ACS. The mean level was 5500 ^ 1400 pg/ml (range 0±13400). The child with the most severe presentation (patient 22) requiring intubation and mechanical ventilation had the highest BALF concentration of IL-8 at 180 000 pg/ml and is not included in the calculation of the mean. These levels were high compared with levels described in healthy adult controls by others [21´5 pg/ml, Nocker et al, (1996); 59´5 pg/ml, Krarup et al, (1997)]. Three children had undetectable

Serum IL-8 Serum IL-8 levels were considerably elevated in all six samples from five adults with ACS and 8 out of 14 children with ACS. Thus, serum IL-8 levels were elevated in 14 and undetectable in 6 out of 20 episodes of ACS. Serum levels of IL-8 were undetectable in all of the 30 control patients with SCD. The number of patients with detectable serum levels of IL-8 was significantly higher among patients with ACS than in the control group (P , 0´0001 by Fisher's exact test). The mean serum concentration of IL-8 was 6500 ^ 3900 pg/ml (range 0±77 467 pg/ml). The mean serum IL-8 level was significantly higher in patients with ACS than the control group (P 0´04). Follow-up serum samples were available in 10 of the 14 episodes in which IL-8 was originally elevated. IL-8 was undetectable after resolution of eight episodes and significantly decreased after two episodes. One patient who developed ACS during a hospital admission for a pain crisis had serial measurements of serum IL-8 levels (Fig 1). These were initially undetectable, increased after the diagnosis of ACS and returned to baseline after resolution of ACS.

Fig 1. Serial measurement of serum IL-8 in a patient. This patient developed ACS during a hospital admission for pain crisis. Serum IL-8 levels were initially undetectable, increased after the diagnosis of ACS, peaked when the patient developed fever and respiratory distress, and subsequently returned to baseline. q 2000 Blackwell Science Ltd, British Journal of Haematology 111: 482±490

IL-8 and G-CSF Levels in Patients with SCD and ACS

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11 patients. The mean IL-8 level was, however, significantly lower that in patients with acute chest syndrome (1900 ^ 740 pg/ml and 5500 ^ 1400 pg/ml respectively; P 0´03). Serum IL-8 levels were undetectable in the 11 patients studied before and during surgery but became detectable in three out of seven of these patients studied 12±24 h after surgery. The mean level of IL-8 in these patients was 400 ^ 270 pg/ml (Table III). Serum levels of IL-8 were undetectable in any of the eight samples obtained in patients without sickle cell disease admitted for severe pneumonia. Serum IL-8 levels and correlation with clinical parameters There was a negative correlation between serum IL-8 levels and the lowest PaO2, which, however, did not reach statistical significance. The relationship between serum IL8 and the alveolar±arterial oxygen tension gradient (A±a) Po2 was also not significant. Fig 2. IL-8 gene expression in a patient with ACS. RNA was isolated from macrophages obtained through bronchoalveolar lavage. Lane 1 contains the 100-bp DNA ladder obtained from Gibco BRL. Lane 2 contains a PCR product from human actin DNA approximately 314 bp in size. Lanes 3±6 contain increasing concentrations of a PCR product from IL-8 DNA approximately 250 bp in size.

serum IL-8 levels but had high BALF levels. One child had high serum, but undetectable BALF levels. Three children had high serum and BALF levels and three children had high BALF levels, but no concomitant serum levels were available. The patient with the highest BALF level of IL-8 had the most severe clinical picture but had only modest levels of circulating IL-8 (100 pg/ml), which became undetectable after exchange transfusion. IL-8 gene expression was demonstrated in the bronchial macrophages of two patients studied (Fig 2), indicating that these cells may have been one of the sources of BALF IL-8. G-CSF levels were elevated in five out of six samples of BALF from children with ACS (mean 2000 ^ 1600 pg/ml, range 0±10300 pg/ml). Serum levels of G-CSF were undetectable in these patients. Patients undergoing surgery and patients with pneumonia Among patients with SCD undergoing surgery, IL-8 was detectable in the endotracheal lavage fluid in 10 out of

Duffy blood group Nineteen of the 22 patients with acute chest syndrome had extended red blood cell phenotypes on record. Only one of the 20 patients (5´2%) was Duffy positive (a2b1). Among the controls, 24 had extended phenotypes and six subjects (25%) were Duffy positive (five Fy a2b1, one Fy a1b2). In the general sickle cell clinic population at MUSC, 57 out of 359 patients phenotyped (16%) were Duffy positive. DISCUSSION In this study we have demonstrated that both BALF and circulating levels of IL-8 were elevated in patients with SCD who developed ACS because of different causes (Table II). Circulating levels of IL-8 could not, however, be correlated with clinical severity of the ACS or response to transfusions. The reason for this may be that the clinical severity of ACS is better correlated with the local production of IL-8 in the lung than with circulating levels of this cytokine. G-CSF levels were also found to be elevated in five out of six samples of BALF but not in serum from children with ACS. These findings suggest that this cytokine was also produced locally in the lung. All of the patients with high BALF G-CSF also had elevated BALF IL-8 levels and pleural effusions. G-CSF has been shown to be produced during acute lung injury in various settings (Tazi et al, 1991; Terashima et al, 1994). In animal models, G-CSF administration exacerbates

Table III. Mean serum levels of IL-8.

n IL-8 level (pg/ml ^ SEM)

Control

ACS

Pre-op

Intra-op

Post-op

Pneumonia

32 0 ^ 0*

20 6500 ^ 1900*

11 0^0

11 0^0

7 400 ^ 270

4 0^0

Mean serum levels of IL-8 in control patients with SCD, patients with acute chest syndrome (ACS), sickle cell patients undergoing surgery under general anaesthesia and four children who did not have sickle cell disease, but were admitted to the intensive care unit because of severe pneumonia.*P 0´02. q 2000 Blackwell Science Ltd, British Journal of Haematology 111: 482±490

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acute lung injury owing to endotoxin and increases extraalveolar lung water (Sakuma et al, 1994; Terashima et al, 1994; King et al, 1995). In a similar observation, a patient with sickle cell disease developed ACS after exogenous administration of G-CSF (Abboud et al, 1998). We have also demonstrated that patients with ACS have pulmonary oedema and vasoocclusion (Bhalla et al, 1993). These findings may be explained in part by increased cytokine production, which leads to increased vascular permeability as well as increased adherence of the endothelium and red cells. IL-8 levels are elevated in the sputum and/or BALF of patients with many lung diseases, including adult respiratory distress syndrome (Lynch et al, 1992; Grunewald et al, 1993; Lipschik et al, 1993; Sekido et al, 1993; Southcott et al, 1995; Nocker et al, 1996; Bohnet et al, 1997; Takizawa et al, 1997). In vitro studies demonstrated that exposure to IL-8 increases the adherence of sickle erythrocytes to endothelium, but no in vivo role for IL-8 has been described in the context of SCD (Kumar et al, 1996). Thus, increased production of IL-8 and other cytokines may explain the development and rapid progression of ACS in response to different initial insults, as has been described in acute respiratory distress syndrome (ARDS). There is also a potential roles for cytokines in the development of chronic lung disease. In neonates with infant respiratory distress syndrome, high levels of plasma IL-8 have been found to correlate with the later development of chronic lung disease (Little et al, 1995). The production of IL-8 during recurrent episodes of ACS could lead to increased sickle erythrocyte adherence (Kumar et al, 1996), neutrophil activation and chronic tissue damage and fibrosis in the lung (Powars et al, 1988). High circulating levels of chemokines, the increased adherence of red cells and high leucocyte numbers could, in turn, also be responsible for the increased risks of mortality (Castro et al, 1994; Platt et al, 1994) and stroke (Frempong et al, 1998) associated with repeated episodes of ACS. The initial stimulus for IL-8 production in these patients is not clear. Hypoxia directly induces IL-8 expression in endothelial cells (Karakurum et al, 1994). Hypoxia, as a result of atelectasis, vasoocclusion or fat embolization, would be followed by activation and recruitment of neutrophils into the lung tissue and alveolar spaces (Baggiolini et al, 1994). Surgery under general anaesthesia and painful vasoocclusive crises (VOC) are associated with the development of ACS (Vichinsky et al, 1995). In this study, we found elevated IL-8 levels in endotracheal lavage fluid and serum of patients who underwent general anaesthesia. It is interesting to note that three of the surgical patients also had elevation of serum secretory phospholipase A2 (PLA2) (data not shown), an enzyme that plays an important role in the pathogenesis of ACS (Styles et al, 1996). However, none of these patients, all of whom had been transfused preoperatively, developed ACS. We did not find elevated IL-8 levels in patients with uncomplicated VOC, but others have described low circulating levels of IL-8 in patients with SCD who had vaso-occlusive crises associated with infection or dehydration (Duits et al, 1998). Inflammatory cytokines such as TNF-a or IL-1b released

in response to infectious or other stimuli also have the ability to induce IL-8 production (Fujishima et al, 1992; Tilg et al, 1993). Even though we could not demonstrate elevated serum levels of TNF-a or IL-1b in patients with ACS, these factors may have been active locally in the lung. Furthermore, exposure of activated neutrophils to IL-8 leads to an increased production of lipid mediators of inflammation, suggesting that IL-8 stimulates the activity of phospholipase A2 (Lipschik et al, 1993; McDonald et al, 1993). PLA2 activation could lead to further IL-8 production (Meier et al, 1995). Thus, induction of IL-8 production in the lung may be a critical common step through which different insults predispose patients, most of whom are Duffy negative, to the development of acute chest syndrome. To date, hydroxyurea is the only drug known to decrease the incidence of ACS (Charache et al, 1995). A potentially significant implication of our findings is that agents that inhibit specific chemokine gene expression, release (Meier et al, 1995; Oishi et al, 1994) or effect on cell adherence may help in the prevention and treatment of ACS. ACKNOWLEDGMENTS The authors would like to thank Dr Elliot Vichinsky for allowing data produced as part of the National Acute Chest Study to be used in this publication. The authors also acknowledge Dr Charles P. Darby and the Children's Hospital Fund at the Medical University of South Carolina for continued support of sickle cell research. REFERENCES Abboud, M., Laver, J. & Blau, C.A. (1998) Granulocytosis causing sickle-cell crisis. Lancet, 351, 609. Baggiolini, M., MoSeries, B. & Clark-Lewis, I. (1994) Interleukin-8 and related chemotactic cytokines (The Giles Filley Lecture). Chest, 105, 95s±98s. Benfield, T., Van Steenvuk, R., Nielsen, T., Dichter, J., Lipschik, G., Jensen, B., Junge, J., Shelhamer, J. & Lundgren, J. (1995) Interleukin-8 and eicosanoid production in the lung during moderate to severe Pneumocystis carinii pneumonia in AIDS: a role of interleukin-8 in the pathogenesis of P. carinii pneumonia. Respiratory Medicine, 89, 285±290. Bhalla, M., Abboud, M.M., CcLoud, T., Shepard, J., Munden, M., Jackson, S., Beaty, J. & Laver, J. (1993) Acute chest syndrome in sickle cell disease: CT evidence of microvascular occlusion. Radiology, 187, 45±49. Bohnet, S., KoÈtschau, U., Braun, J. & Dalhoff, K. (1997) Role of interleukin-8 in community-acquired pneumonia: relation to microbial load and pulmonary function. Infection, 25, 95±100. Castro, O., Branbilla, D., Thorington, B., Reindorf, J., Gillette, P., Vera, J. & Levy, P. (1994) The Cooperative Study of Sickle Cell Disease: the acute chest syndrome in sickle cell disease: incidence and risk factors. Blood, 84, 643±649. Charache, S., Terrin, M.L., Moore, R.D., Dover, G.J., Barton, F.B., Eckert, S.V., McMahon, R.P. & Bonds, D. (1995) Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. New England Journal of Medicine, 332, 1317±1322. Chollet-Martin, S., Jourdain, B., Gibert, C., Elbim, C., Chastre, J. & Gougerot-Pocidalo, M. (1996) Interactions between neutrophils and cytokines in blood and alveolar spaces during ARDS.


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