Alveolar macrophages and CC chemokines are increased in children with cystic fibrosis.
Siobhain Brennan
1,2
, Peter D. Sly
1,2,3
, Catherine L Gangell
1,2
, Nina Sturges
1,2
, Kaye
Winfield 1,2 , Matt Wikstrom 1,2 , Samantha Gard 1,2 , and John W. Upham 1,2,4 on behalf of
AREST CF
2,5
.
1 Telethon Institute for Child Health Research, Perth, Australia.
2 Centre for Child Health Research, University of Western Australia, Perth, Australia.
3 Department of Respiratory Medicine, Princess Margaret Hospital for Children,
Perth, Australia.
4 School of Medicine, University of Queensland, Brisbane, Australia.
5 Murdoch Children’s Research Institute, Melbourne, Australia.
Address for correspondence:
Professor Peter Sly
Division of Clinical Sciences,
Telethon Institute for Child Health Research,
P.O. Box 855, West Perth
W.A. 6872, Australia
Email: peters@ichr.uwa.edu.au
Phone: 61+ 8+ 9489 7814
Fax: 61+ 8+ 9489 7700
1

Abbreviations
ANOVA
AM
BAL
CC chemokine
CCL
CD
CF
CFTR
CFU
DC
EDTA
ELISA
ERS
FITC
GRO
HLA
IL
MCP
MIP
NE
NF-
NK

B
RANTES
TNF analysis of variance alveolar macrophage bronchoalveolar lavage chemokines with two adjacent cysteines near their amino terminus
CC chemokine ligand cluster of differentiation cystic fibrosis cystic fibrosis transmembrane conductance regulator colony forming units dendritic cell ethylenediaminetetraacetic acid enzyme-linked immunosorbent assay
European Respiratory Society fluorescein isothiocyanate growth regulated oncogene human leukocyte antigen interleukin monocyte chemoattractant protein macrophage inhibitory protein neutrophil elastase nuclear factor kappa B natural killer regulated upon activation, normal T-cell expressed and secreted tumour necrosis factor
2

METHODOLOGY
AREST CF Annual Surveillance Program.
This program was established at Princess Margaret Hospital for Children, Perth,
Australia in 1999 when it was recognised that bacterial organisms (including
Pseudomonas species) and evidence of early inflammation (with or without infection) were being detected in significant numbers of CF patients from BAL specimens taken as early as 6 weeks of age. The program was extended to the Royal Children’s
Hospital, Melbourne Australia in 2005. Children are diagnosed with CF following detection by newborn screening and a BAL is now performed on all children diagnosed with CF in Western Australia and in Victoria (with the exception of the southern metropolitan region) according to the following schedule:

Following diagnosis (usually ~ 12 weeks of age)

Annually after that until 6 years old or able to produce sputum voluntarily

At other times if clinically indicated

In addition, if Pseudomonas species are detected, a BAL is repeated one month after eradication treatment is completed to assess for clearance of the organism.
The surveillance program also includes measurement of lung function using infant or preschool techniques, chest CT scanning and assessment of inflammatory markers in
BAL and urine.
As a result of the BAL program, a treatment regime was developed for patients who returned a positive culture for Pseudomonas from BAL is as follows:
 patient admitted for a 2 week course of intravenous anti-pseudomonal antibiotics
(Ticarcillin/Clavulante and Tobramicin unless otherwise indicated).
3

 if good clinical response, discharged home on oral Ciprofloxacin and nebulised
Tobramycin for a month before reverting to regular antibiotic prophylaxis.
 re-admitted for repeat BAL a further month later to assess for clearance of the
Pseudomonas species.
All BAL specimens are assessed for microbiology culture (including virology immuno-fluorescence, PCR and /or culture as appropriate) inflammatory cells and inflammatory markers with if clinically indicated.
BAL procedure
Flexible bronchoscopy was performed via a laryngeal mask under general anaesthesia using intravenous propofol (3-4 mg/kg), following pre-medication with midazolam
(0.5mg/kg) if required. Suction through the bronchoscope was delayed until the tip passed the carina. With the bronchoscope wedged into the right middle lobe bronchus three separate aliquots of normal saline (1ml/kg) were instilled and retrieved.
The first aliquot retrieved is sent for identification of microbes, fungal elements and viruses. The second and third aliquots collected are pooled and stored on ice until processed (within 3 hours) for the assessment of inflammation.
Processing of BAL Fluid
The pooled samples were centrifuged (167xg for 5 mins) and the cell free supernatant decanted, aliquoted, and frozen at –80 o C for later analysis. The cell pellet was resuspended to 10
6
cells/ml and a total cell count (TCC) conducted using duplicate cytospins and Leishman’s staining. A differential cell count of 300 cells was then conducted.
4

The same BAL procedure was followed for NCF subjects undergoing clinical investigation for unresolved respiratory symptoms.
Microbiological Assessment:
BAL samples were cultured on Blood, CLED, Fildes agar and Sarabouds agar with chloramphenicol. P. aeruginosa were isolated from Horse Blood Agar and Horse
Blood and Ticarcillin agar plates incubated in a 5% CO
2
atmosphere at 34 degrees centigrade for 48 to 72 hours and colonies described as mucoid, smooth or rough by their appearance on Horse Blood Agar. Significant microbial infection was considered as a bacterial density of >10
4 cfu/ml and isolated colonies recorded at <10
4
cfu/ml.
BAL samples that cultured mixed oral flora and colonies <10
4
cfu/ml were classified as uninfected.
5

RESULTS
Table E1 . Chemokine levels of children with CF with negative BAL culture compared with Non-CF controls with no defined pathogenic infection
Non-CF CF subjects
Number
Age (yrs)
8
3.48 (1.8, 5.15)
12
2.1 (0.64, 3.16)
12
BAL Culture Results
Negative culture 0
Mixed oral flora only 8
Mixed oral flora in co-culture (2 nd 2 organisms isolated colonies only)
Isolated colonies of candida spp. 1
Isolated colonies of H. influenzae 1
MCP-1 (CCL2) levels in BAL pgmL
-1
26.25 (16.3, 29.4)
MIP-1

(CCL3) levels in BAL pgmL
-1 4.3 (3.5, 6)
Mip-1

(CCL4) levels in BAL pgmL -1 24.7 (19.6, 34.5)
Mip-3

(CCL20) levels in BAL pgmL
-1 33.0(22.7, 85.8)
RANTES (CCL5) levels in BAL pgmL
-1
21.0 (10.3, 47.8)
* P=<0.05
251.4 (100.2, 354. 3)*
31.2 (23.8, 62.0)*
218.0 (83.4, 370.6) *
192.9 (116. 2, 296.5)*
12.05 (3.2, 25.2)
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Table E2: Clinical features of CF and Non-CF subjects related to infection status.
Parameters n
Non-CF uninfected
15
Non-CF infected
9
CF uninfected
CF isolated colonies and MOF
¶
12 23
CF infected
16 p

Age (years)
Weight (kg)
3.52
(2.19, 5.57)
1.82
(1.59, 2.88)
3.07
(2.61, 3.53)
2.22
(0.78, 3.38)
3.68
(0.43, 4.97)
18.6
(14.5, 21.8)
13.9
(9.0, 17.2)
14.1
(13.3, 16.3)
12.8
(9.9, 14.9)
15.3
(6.8, 17.7)
0.41
0.11
BAL return (%)
Previous BAL infection [n (%)]*
28.5
(14.9, 40.2)
18.6
(10.7, 27.8)
33.8
(28.9, 45.3)
33.1
(25.9, 40.3)
31.7
(22.1, 36.7)
4/12
(33%)
9/15
(60%)
6/9
(67%)
0.24
Respiratory symptoms [n (%)]
9
(75%)
9
(100%)
2
(17%)
7
(30%)
4
(25%)
0.012
Data presented as median (IQR);
¶
Isolated colonies defined as infection <10 4 cfu/ml, MOF=mixed oral flora;

Comparison between non-CF uninfected and CF uninfected. Non-parametric Wilcoxon Rank
Sum Test used for continuous variables and Fisher’s exact test used for binary variables; *If first BAL then excluded from this analysis.
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Table E3 : Chemokine levels in BAL fluid in children with CF less than 18 months of age with negative BAL culture compared with Non-CF controls
Non-CF CF
Number
Age (yrs)
6
4.6 (3.5, 5.5)
BAL Culture Results
Negative culture
Mixed oral flora only 6
MCP-1 levels in BAL pgmL
-1
20.9(15.4, 27.9)
MIP-1

levels in BAL pgmL
-1 3.6 (3.4, 5.0)
Mip-1

levels in BAL pgmL -1 24.3(15.3, 24.7)
Mip-3

levels in BAL pgmL
-1 26.1(20.6, 86.1)
RANTES levels in BAL pgmL
-1
27.3 (9.7, 56.1)
*p<0.05
0
5
1.1 (0.2, 1.1)
5
0
127.4(94.6, 412. 6)*
34.5, (24.8, 73.7)*
176.1(154.5, 478.2)*
222.8 (111.2, 406.3)*
17.1 (10.7, 41.9)
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Table E4: Correlation between chemokines in BAL, BAL Inflammatory cells and blood DC subsets in subjects with CF (n=51). Pearson correlation coefficients (r) and p value are shown, together with the numbers of subjects (n) contributing data to the analyses.
MCP-1 r p n
MIP-1
 r p n
MIP-1
 r p n
MIP-3
 r p n
IL-8 r p n
RANTES r p n
0.44
0.001
51
0.32
0.03
48
0.46
0.000
51
0.20
0.16
50
TCC/mL x10 6
0.73
0.0001
51
0.46
0.000
51
0.44
0.001
51
0.53
0.000
48
0.76
0.000
51
0.30
0.04
50
Neutrophil
/mLx10 3
0.48
0.0001
51
0.54
0.000
51
0.33
0.019
51
0.18
0.21
48
0.11
0.42
51
0.11
0.43
50
Macrophage/ mLx10 3
0.33
0.019
51
0.33
0.02
51
IL-6
0.55
0.0001
51
0.55
0.0001
51
0.56
0.0001
51
0.61
0.0001
48
0.38
0.006
50
9

Legends for Figures
Figure E1 : Numbers of macrophages and neutrophils in BAL obtained from children with cystic fibrosis with no infection (  ), isolated colonies (  ) or a current infection
(

) Cell numbers are expressed as number x10
3
/ mL fluid retrieved and have been log transformed. A significant relationship between the number of BAL macrophages and neutrophils was seen. [r=0.42, p=0.0004].
Figure E2 : Numbers of macrophages and neutrophils in BAL obtained from children with cystic fibrosis with no infection (  ), isolated colonies (  ) or a current infection
(

) Cell numbers are expressed as number x10
3
/ mL fluid retrieved and have been log transformed. A significant relationship between the number of BAL macrophages and neutrophils was seen. [r=0.66, p=0.0006].
Figure E3a : BAL levels of MCP-1 in children with (  ) or without (

) cystic fibrosis in the absence (open symbols) or presence (closed symbols) of a pulmonary infection.
Figure E3b : BAL levels of MIP-1

in children with (  ) or without (

) cystic fibrosis in the absence (open symbols) or presence (closed symbols) of a pulmonary infection.
Figure E3c : BAL levels of MIP-1

in children with (  ) or without (

) cystic fibrosis in the absence (open symbols) or presence (closed symbols) of a pulmonary infection.
Figure E3d : BAL levels of RANTES in children with (  ) or without (

) cystic fibrosis in the absence (open symbols) or presence (closed symbols) of a pulmonary infection.
Figure E3e : BAL levels of MIP-3

in children with (  ) or without (

) cystic fibrosis in the absence (open symbols) or presence (closed symbols) of a pulmonary infection.
Figure E4 : BAL levels of IL-8 in children with (  ) or without (

) cystic fibrosis in the absence (open symbols) or presence (closed symbols) of an infection.
Figure E5 : BAL levels of IL-6 in children with (  ) or without (

) cystic fibrosis in the absence (open symbols) or presence (closed symbols) of an infection.
10