Eosinophil-Associated

Lung Diseases :

A Cry for Surfactant Proteins A

and D Help?

Gamal Rabie Agmy, MD, FCCP Professor of chest Diseases, Assiut university

Eosinophil-Associated Lung Diseases

A Cry for Surfactant Proteins A and D

Help?

Julie G. Ledford, Kenneth J. Addison, Matthew W. Foster, and

Loretta G. Que

American Journal of Respiratory Cell and Molecular Biology Volume 51 Number

5 | November 2014

Clinical Relevance

This lecture summarizes the functions of surfactant

proteins A and D (SP-A/-D) in the lung and proposes

several mechanisms by which SP-A/-D may regulate

eosinophil activity and function in inflammatory airway

diseases.

Understanding how SP-A/-D regulate airway inflammation

will help us to develop novel strategies to treat lung

diseases in which eosinophils play an important role in

disease pathogenesis.

Surfactant proteins (SP-A, SP-B, SP-C, and SP-D) make

up 5 to 10% of all pulmonary surfactant. They differ from

one another in their synthesis, oligomerization, and

function.

Whereas SP-B and SP-C are hydrophobic and function to

prevent alveolar collapse by reducing surface tension in

the distal lung, SP-A and SP-D are hydrophilic and play

unique and important roles in lung host defence.

Altered SP-A/-D levels are commonly observed as

markers of airway and lung diseases. However, the

clinical associations with disease are ambiguous as to

whether SP-A/-D dysregulation/dysfunction is important

for disease pathology or rather a byproduct of the

diseased lung environment.

In this lecture we highlight the association of altered

surfactant protein levels in eosinophil-dominated lung

diseases and speculate on possible mechanisms by

which SP-A/-D may regulate or be regulated by

eosinophil functions, thus highlighting a lesser

appreciated area of SP-A/-D immunobiology.

Roles of SP-A/-D in

Immunity

An insight into the diverse roles of

surfactant proteins, SP-A and SP-D in

innate and adaptive immunity

Front. Immunol., 07 June 2012

Roles of SP-A/-D in Immunity Surfactant proteins SP-A and SP-D are hydrophilic,

collagen-containing calcium-dependent lectins, which

appear to have a range of innate immune functions at

pulmonary as well as extrapulmonary sites.

These proteins bind to target ligands on pathogens,

allergens, and apoptotic cells, via C-terminal homotrimeric

carbohydrate recognition domains, while the collagen

region brings about the effector functions via its

interaction with cell surface receptors.

SP-A and SP-D deal with various pathogens, using a range

of innate immune mechanisms such as

agglutination/aggregation, enhancement of phagocytosis,

and killing mechanisms by phagocytic cells and direct

growth inhibition.

Roles of SP-A/-D in Immunity

SP-A and SP-D have also been shown to be involved in the

control of pulmonary inflammation including allergy and

asthma.

Emerging evidence suggest that SP-A and SP-D are

capable of linking innate immunity with adaptive immunity

that includes modulation of dendritic cell function and

helper T cell polarization.

Metabolism of SP-A/-D in

the Normal Lung

The majority of SP-A is synthesized by alveolar type (AT)II cells in

the distal airways. Newly synthesized SP-A is packaged into

lamellar bodies by the ATII cells for storage and ensuing secretion

via regulated exocytosis.SP-A is not only synthesized and secreted

from the ATII cells; it is also predominantly recycled by them.

Although SP-D is synthesized and secreted constitutively by ATII

cells, it is also produced by nonciliated Clara cells in the upper

airways .Similar to SP-A, SP-D release is regulated by granule

exocytosis . Although the majority of surfactant phospholipid and

protein are removed from the alveolus by ATII cell uptake , evidence

suggests that alveolar macrophages are also important participants

in the uptake and degradation of exhausted surfactant protein.

Uptake by macrophages and ATII cells is mediated predominantly

by endocytosis via clathin-coated pits

Asthma Based on studies in murine models of allergic

inflammation and in studies of patients with asthma, it

has become increasingly evident that SP-A and SP-D play

important regulatory roles in allergic airways diseases.

Mice challenged with house dust mite (HDM), or fungi

have alterations in SP-A/-D levels at the height of

eosinophilia

An increase in SP-A/-D during eosinophilic inflammation

is likely a key defense mechanism for eosinophil

regulation: SP-D inhibits eosinophil chemotaxis, SP-A

and SP-D bind eosinophils and attenuate degranulation,

and SP-A suppresses IL-8 production from eosinophils .

Asthma In support of these findings, when mice lacking SP-A or

SPD are challenged with allergen, they develop severely

enhanced eosinophilia compared with WT control mice and

display worsened symptoms of allergic airways

inflammation tied to Th2- dominant disease

In studies of segmental antigen challenge in atopic and

individuals with asthma, the ability of total surfactant

(lipoproteins and proteins) extracted from the antigen-

challenged lobe was found to be dysfunctional in its ability

to maintain airway patency as compared with surfactant

from a control (saline challenged) lobe

Asthma Normal activity could be achieved by removing water-

soluble inhibitors from the extracted surfactant, which was

attributed to leakage of plasma proteins into the lumen

during inflammation.Studies have demonstrated that

plasma proteins, albumin, and fibrinogen impair surfactant

function at physiological concentrations

Additionally, a product released from activated

eosinophils, eosinophil cationic protein, had a profound

effect on the arrangement of phospholipids within the

surfactant biofilm due to what appeared to be unwinding of

the lamellar bodies . Normally, SP-A will partially protect

from this surfactant-inhibiting effect. However, in chronic

inflammatory lung conditions such as asthma, functional

SP-A levels may be decreased and unable to adequately

regulate this interaction.

Although the mechanisms for asthma-derived SP-A

dysfunction are unclear, the increased eosinophilia and

eosinophil-derived factors (eosinophil peroxidase,

eosinophil cationic protein, eosinophil associated RNase,

and major basic protein) associated with Th2-

predominant asthma may alter SP-A oligomerization and

render SP-A incapable of carrying out normal host

protective functions.

Asthma

Allergic Bronchopulmonary

Aspergillosis

The ability of SP-A and SP-D to interact with the

glycosylated antigens and allergens of the fungal

pathogen Aspergilous fumigatus (AFU) to inhibit specific

IgE binding to these allergens makes them an attractive

therapeutic target for AFU-associated diseases

Murine models of pulmonary hypersensitivity induced by

AFU immunologically resemble the human disease

allergic bronchopulmonary aspergillosis (ABPA) a

condition characterized by serum and pulmonary

eosinophilia, hypersensitivity to AFU, and increased total

IgE. These models have successfully established a

protective role for SP-A and SP-D in the treatment of

ABPA.

Expression of SP-D is increased in BAL in AFU

sensitized mice and in serum of humans with ABPA .

Ablation of SP-A and SP-D leads to enhanced

eosinophilia and increases total IgE in AFU-sensitized

mice

treatment of AFU-sensitized WT and SP-A– and SP-D–

deficient mice with “rescue” SP-A and SP-D,

respectively, has been shown to suppress IgE

levels,eosinophilia, cellular inflammation in the lung,

and shift the pathogenic TH2 cytokine profile to a

protective TH1 profile

Allergic Bronchopulmonary

Aspergillosis

Surfactant protein D in serum from patients

with allergic bronchopulmonary aspergillosis

Serum SP-D in CF patients was significantly higher than in the controls

without lung disease .

During the whole ABPA episode, SP-D level did not change significantly,

despite large changes of total serum immunoglobulin E.

There was a clear negative correlation between SP-D concentration and

overall lung function, i.e. forced expiratory volume in one second and

forced vital capacity.

Serum level of surfactant protein D may be of value to follow pulmonary

function and lung injury in cystic fibrosis patients.

Surfactant protein D serum levels are not helpful for the diagnosis and

follow-up of an allergic bronchopulmonary aspergillosis episode,

contrary to what was expected from animal experiments.

Eur Respir J 2003; 22: 592–595.

Acute Eosinophilic Pneumonia

Acute eosinophilic pneumonia (AEP) is a rare disease of

unknown etiology characterized by acute respiratory

failure, bilateral infiltrates,and eosinophilic infiltration of

the lung .

Unfortunately, no animal models for AEP exist. Although

the pathophysiology of AEP is unknown, eosinophils are

believed to play a role because they comprise greater

than 25% of BAL cells and because IL-5 and IL-1ra are

detected at increased levels in the BAL of affected

patients .Levels of SP-A and SP-D in BAL and serum are

reported to be significantly elevated in patients with AEP

compared with healthy control subjects

It is not clear what the implications of these

observations are for the pathobiology of AEP.

One may speculate that, during AEP, the increase in

eosinophilia would lead to SP-A/-D breakdown and

dysfunction, and therefore, as a compensatory

mechanism, more SP-A/-D would be produced and

secreted in an attempt to regulate eosinophil activities.

Acute Eosinophilic Pneumonia

Allergic Rhinitis

Allergic rhinitis (AR) is an IgE-mediated chronic

inflammatory disease characterized by the recruitment of

eosinophils, basophils, and T cells expressing TH2

cytokines to the nasal mucosa .

In a murine model of AR, treatment with exogenous SP-

A decreased eosinophil number in nasal epithelium,

corrected the TH1/TH2 imbalance, and blocked

ovalbumin (OVA)-specific IgE ; these findings strongly

suggest a protective role for SP-A in AR.

In humans, SP-A, -B, -C, and –D are components of

healthy nasal mucosa and have been shown to increase

with inflammation, with the exception of SPC

Expression of SP-A is much higher in patients with AR

and nasal polyps than in control subjects, and the level

of SP-A positively correlates with eosinophil number

within the basement membrane of epithelium

To date, only SP-A expression in nasal mucosa has been

shown to correlate with severity of disease as measured

by the Rhinitis Symptom Utility Index in patients with AR

,suggesting that it plays a key role in the inflammatory

process regulating AR and nasal polyp formation in

these patients.

Allergic Rhinitis

Interstitial Lung Disease

Elevated serum SP-A levels have been reported in patients

diagnosed with HP and idiopathic pulmonary fibrosis (IPF),

however, SP-A levels are not consistently elevated in the

BAL of these patients

Findings in IPF show a significant negative correlation

between BAL SP-A levels and the presence of BAL

eosinophils. Low levels of BAL SP-A in subjects with

enhanced eosinophilia associated with IPF suggest that

SP-A may be involved in the regulation of eosinophil

recruitment, survival, or resolution in the lung in response

to environmental stresses.

An alternative explanation could be that in patients with

IPF with associated eosinophilia, degradation of SP-A

occurs, lowering the detectable levels of SP-A.

COPD

COPD is generally considered to be a neutrophilic

disease. However, there is increasing evidence to suggest

that a subgroup of patients with stable COPD exists that

have chronic airway eosinophilia and steroid responsive

Disease

Decreased levels of BAL SP-A and SP-D have been

detected in healthy smokers compared with nonsmoking

control subjects. The decreased concentration of SP-A

and SP-D in lung lavage in smokers is speculated to

impair the host defense functions of surfactant in the

peripheral airways and may contribute to the development

of chronic obstructive lung disease.

In patients with COPD, sputum SP-A/-D and serum SP-

D levels associate with lung function and with health

status and increase significantly during COPD

exacerbations , suggesting that SPD may be a

biomarker of disease severity for COPD

The Real Story

These data are communicated for scientific purpose only. Confidential slide set 28

Apoptotic Pathways in COPD

Demedts IK, et al. Respir Res. 2006;7:53. Reproduced with permission from Biomed Central.

Survival factor Granzyme B Perforin

TNF-α sFasL

cytoplasm

nucleus

ER Stress

Apoptosome

Apaf 1 Procasp-9

Procasp-9 Casp-9

Casp-8 CAD CAD

ICAD

Casp-8

Procasp-8 Procasp-8

FADD Bid tBid

Bax

Bak

Cyt C

ER stress

DNA fragmentation

1 2

4

3

5

?

Fas

COPD Pathogenesis

These data are communicated for scientific purpose only. Confidential slide set 30

Angiogenesis in COPD

Reprinted f rom International Journal of COPD, 2, Siafakas NM, et al., Role of angiogenesis and vascular remodeling in

chronic obstructive pulmonary disease, 453-462, Copyright 2007, with permission f rom Dove Medical Press Ltd.

extravasated

plasma proteins

Inflammatory cells (Mac, Neu, Epith, Lymph)

Release of angiogenic

mediators

Fibrinogen products

Inflammation Tissue

hypoxia

Airway

fibrosis

Mechanical

Injury

Increased

blood flow

Vessel growth

Angiogenesis

Vascular remodeling

Up-regulation of

Angiogenic factors

Shear stress

on the endothelium

COPD Pathogenesis

These data are communicated for scientific purpose only. Confidential slide set

Angiogenic and Angiostatic Factors in COPD

• Angiogenic CXC Chemokines, CC Chemokines, and Growth Factors:

– CXCL1

– CXCL5

– CXCL8

– CCL2

– VEGF

– bFGF

– Angiopoietin-1

– HGF

– EGF

• Angiostatic CXC Chemokines, CC Chemokines, and Growth Factors:

– CXCL10

– CXCL11

Siafakas NM, et al. Int J Chron Obstruct Pulmon Dis. 2007;2:453-462.

COPD Pathogenesis

Global Strategy for Diagnosis, Management and Prevention of COPD

Definition of COPD

◙ COPD, a common preventable and treatable

disease, is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases.

◙ Exacerbations and comorbidities contribute to the overall severity in individual patients.

GOLD 2014

These data are communicated for scientific purpose only. Confidential slide set 33

Inflammatory Mediators in COPD – Summary

Cell

Neutrophils

Macrophages

T-cell

Epithelial cell

IL-8, TGF- 1, IP-10, Mig, I-TAC, LTB4, GRO- , MCP-1, MMP-9

Granzyme B, perforins, IFN-, TNF-

IL-8, IL-6, TGF-1 TGF-, IP-10, Mig, I-TAC, LTB4, GRO-, MCP-1, ROS, MMP-9

Serine proteases, TNF-, ROS, IL-8, MPO, LTB4

Selected Mediators

Barnes PJ, et al. Eur Respir J. 2003;22:672-888.

Inflammation in COPD

Influencing The Cellular Components

Of Inflammation

Phosphodiesterase Inhibitors

The PDE4 isoenzyme is a major therapeutic target

because it is the predominant isoenzyme in the majority

of inflammatory cells, including neutrophils, which are

implicated in the pathogenesis of COPD. Inhibition of

PDE4 in inflammatory cells influences various specific

responses, such as the production and/or release of pro-

inflammatory mediators including cytokines and active

oxygen species , with a well-documented efficacy in

animal models of COPD .

Influencing The Cellular Components

Of Inflammation

Phosphodiesterase Inhibitors

Oral PDE4 inhibitors: roflumilast; GRC-3886;

ELB353; GRC 4039; MEM1414; oglemilast;

OX914; ASP3258; TAS-203; Zl-n-91; NIS-

62949; tetomilast

Inhaled PDE4 inhibitors; GSK256066;

SCH900182; Compound 1; tofimilast;

AWD12-281; UK500001

PDE3/4 inhibitors: RPL554

PDE4/7 inhibitors: TPI 1100

Influencing The Cellular Components

Of Inflammation

Adenosine receptors Agonist

Some evidence suggests the involvement of adenosine

receptors in inflammation. Four subtypes (A1, A2A, A2B, A3) of

adenosine receptors have been characterized. The anti-

inflammatory effect of adenosine is due to a short-term

activation of A2A receptor that elevates cAMP and,

consequently, modulates key pro-inflammatory neutrophil

functions such as superoxide generation, degranulation and

adhesion. Furthermore, adenosine A2A receptor activation

induces a shift in the profile of lipid mediator production from

leukotrienes to prostaglandin E2.This shift may contribute to

prevent the subsequent neutrophil-elicited inflammatory

events

Influencing The Cellular Components

Of Inflammation

Adenosine receptors A2a Agonists

CGS21680; ATL146e; UK371,104; GW328267X;

regadenoson (CVT-3146); 2-(cyclohexylethylthio)-AMP

Influencing The Cellular Components

Of Inflammation

Adhesion molecules Inflammatory processes in COPD are coupled to an increased

recruitment of neutrophils to the lung in response to a release of IL-8

and leukotriene B4 (LTB4) by activated epithelial cells and

macrophages . Migration of inflammatory cells from the vascular

compartment to the surrounding tissue is partly regulated by

selectins (L-, P- and E-selectin) . Selectins mediate transient adhesive

interactions pertinent to inflammation through the recognition of the

carbohydrate epitope, sialyl Lewisx (sLex), expressed on circulating

leukocytes. The rapid turnover of selectin--ligand bonds mediates the

cell tethering and rolling in shear flow. Several studies suggest that

selectins are involved in the inflammatory processes of COPD .

Therefore, targeting these molecules might reduce the inflammation

in COPD

Influencing The Cellular Components

Of Inflammation Drugs that interfere with adhesion molecules

Carbohydrate-based inhibitors: sLex antagonists

(bimosiamose); heparins and heparinoids (PGX-

100, PGX-200); synthetic glycomimetic molecule

(GMI-1070) mAb inhibitors: EL246

Influencing The Inflammatory mediators

1-TNF-a

2-Chemokines

3-NF-kB

4-p38 MAPK and MK2

5-PI3K

6-LTB4

7-PPAR

Targeting protease activity at the

enzymatic level

Drugs that may have indirect anti-

inflammatory actions

Reversing glucocorticoid resistance :

Activation of HDAC2: theophylline;

curcumin; resveratrol

Inhibition of P-glycoprotein

Inhibition of MIF

These data are communicated for scientific purpose only. Confidential slide set

Bronchodilators in COPD

Influencing the bronchial tone

Bronchodilation may be obtained either by

directly relaxing the smooth muscle

through stimulation of the b2-AR with b2-

AR agonists, or/and by inhibiting the

action of ACh at mAChRs.

Bronchodilators

Indacterol Glycopyrronium bromide

Olodaterol Aclidinium bromide

Vilanterol

Xanthines

Influencing the bronchial tone

Inhibitory NANC (iNANC) system is considered to be

the main neural mechanism mediating ASM relaxation

by releasing of vasoactive intestinal peptide (VIP), VIP

structure-related peptides and nitric oxide (NO) .

On the other hand, excitatory NANC (eNANC) system

mediates bronchial contraction activating the efferent

functions of bronchopulmonary-sensitive sensory

nerves. These nerves release tachykinins, such as

substance P and neurokinin A, which in turn activate

neurokinin-1 (NK-1) and NK-2 receptors located on the

ASM membrane, thus inducing bronchoconstriction

Influencing the bronchial tone

Bronchodilation may, therefore, be

obtained either by directly relaxing the

smooth muscle through stimulation of the

b2-AR with b2-AR agonists, or/and by

inhibiting the action of ACh at mAChRs.

Furthermore, an alternative approach

could be the modulation of the NANC

system.

The effectiveness of the serum surfactant protein D

(Sp-D) level to indicate lung injury in pulmonary

embolism.

Surfactant protein D (SP-D) is a biomarker specific to the lungs. Our aim was

to investigate the relationship between clinical probability scores and the

serum levels of SP-D to indicate the severity of lung injury that develops

secondary to hypoxia in pulmonary embolism (PE).

METHODS:

We included three groups in the study: non-massive PE (n = 20), sub-massive

PE (n = 20), and the control group (n = 20), which consisted of healthy

volunteers. The modified Geneva and Wells clinical probability scoring

systems were performed for PE, and the patients were classified as low risk,

moderate risk, and high risk. SP-D levels were determined by the enzyme-

linked immunosorbent assay.

CONCLUSIONS:

In our study, SP-D levels were significantly higher in the sub-massive PE

group overall. However, further prospective studies are required with a larger

number of cases, including patients with massive PE, in order to clarify the

findings 64-1457):9(60;2014 Lab. Clin

Possibility of Therapeutic

Surfactants in Eosinophilic

Diseases

Although surfactant therapy in preterm neonates had

dramatically changed their long-term outcome, the

current formulation is devoid of SP-A and SP-D.

Additionally, preterm neonates often suffer from

respiratory distress syndrome and have a higher risk

for development of infection and bronchopulmonary

dysplasia. Given our current knowledge of the

protective roles of SP-A and SP-D in these areas,

clinicians using new surfactant therapies may consider

the addition of SP in the treatment of these patients.

An animal model using AFU-allergen in SPD–deficient

mice shows that exogenous SP-D treatment given to the

mice can rescue their allergic phenotype.

SP-D fragments were capable of reducing early airway

responses to AFU allergen and led to significantly

decreased airway hyperresponsiveness, eosinophilia,

and histamine levels as compared with placebo

Lung diseases with increased SP-A/-D in BAL

1-Astma

2-AEP

3-LCH

4- HP

5-PAP

Lung diseases with decreased SP-A/-D in BAL

1-Smokers

2-COPD

3-IPF

4- Sarcoidosis

5-IPCD

Conclusions Pulmonary surfactant proteins are critical in mediating a variety

of immune and physiological responses during health and

disease. Many lung diseases associated with eosinophilia also

have dysregulated SP-A/-D metabolism, as detected by altered

levels in serum, BAL, or both. Although the etiology of altered

levels of SP-A/-D is unclear in each of the diseases mentioned,

SP-A and SPD bind eosinophils and regulate their

degranulation.

Surfactant proteins (SP)-A and SP-D (SP-A/-D) play important

roles in numerous eosinophil-dominated diseases, including

asthma, allergic bronchopulmonary aspergillosis, and allergic

rhinitis. In these settings, SP-A/-D have been shown to

modulate eosinophil chemotaxis, inhibit eosinophil mediator

release, and mediate macrophage clearance of apoptotic

eosinophils.

Dysregulation of SP-A/-D function in eosinophil-dominated

diseases is also not uncommon. Alterations in serum SP-A/-

D levels are associated with disease severity in allergic

rhinitis and chronic obstructive pulmonary disease.

Furthermore, oligimerization of SP-A/-D, necessary for their

proper function, can be perturbed by reactive nitrogen

species, which are increased in eosinophilic disease.

Although rescue treatments that give exogenous

full-length or peptides of SP-A or SP-D have

shown promising results in allergic animal

models, to the best of our knowledge, no studies

have examined the therapeutic potential of

purified SP-A/-D or targeted SP-A/-D peptides in

human lung diseases in which eosinophils are

thought to play an important role in

pathogenesis.

Identification and Quantitation of Coding Variants and Isoforms

of Pulmonary Surfactant Protein A.Foster MW, et al. J

Proteome Res, 2014 Jul 15. PMID 25025725,

Establishment of surfactant-associated protein A suicide gene

system and analysis of its activity.Zhang WG, et al. J Huazhong

Univ Sci Technolog Med Sci, 2014 Jun. PMID 24939295

Pilot study exploring lung allograft surfactant protein A (SP-A)

expression in association with lung transplant

outcome.D'Ovidio F, et al. Am J Transplant, 2013 Oct. PMID

24007361

Surfactant protein A suppresses lung cancer progression by

regulating the polarization of tumor-associated

macrophages.Mitsuhashi A, et al. Am J Pathol, 2013 May.

PMID 23499372

Lipoteichoic acid induces surfactant protein-A biosynthesis in

human alveolar type II epithelial cells through activating the

MEK1/2-ERK1/2-NF-κB pathway.