2. Eosinophil-Associated
Lung Diseases :
A Cry for Surfactant Proteins A
and D Help?
Gamal Rabie Agmy, MD, FCCP
Professor of chest Diseases, Assiut university

3. 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

4. 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.

6. 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.

7. 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.

8. Roles of SP-A/-D in
Immunity

9. An insight into the diverse roles of
surfactant proteins, SP-A and SP-D in
innate and adaptive immunity
Front. Immunol., 07 June 2012

10. 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.

11. 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.

12. 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

13. 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 .

14. 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

15. 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.

16. 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

17. 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.

18. 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

19. 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 wholeABPA 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.

20. 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

21. 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

22. 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.

23. 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

24. 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.

25. 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.

26. 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

27. The Real Story

28. 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-8Procasp-8
FADDBidtBid
Bax
Bak
Cyt C
ER
stress
DNAfragmentation
1
2
4
3
5
?
Fas
COPD Pathogenesis

30. These data are communicated for scientific purpose only. Confidential slide set
30
Angiogenesis in COPD
Reprinted from 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 from 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

31. 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:
– CXCL10Siafakas NM, et al. Int J Chron Obstruct Pulmon Dis. 2007;2:453-462.
COPD Pathogenesis

32. 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

33. These data are communicated for scientific purpose only. Confidential slide set
33
Inflammatory Mediators in COPD – Summary
Cell
Neutrophils
Macrophages
T-cell
Epithelialcell
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

34. 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 .

35. 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

36. 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

37. Influencing The Cellular Components
Of Inflammation
Adenosine receptors A2a Agonists
CGS21680; ATL146e; UK371,104; GW328267X;
regadenoson (CVT-3146); 2-(cyclohexylethylthio)-AMP

38. Influencing The Cellular Components
Of Inflammation
Adhesion molecules
Inflammatory processes in COPD are coupled to an increased
recruitmentof neutrophilsto 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

39. 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

40. Influencing The Inflammatory mediators
1-TNF-a
2-Chemokines
3-NF-kB
4-p38 MAPK and MK2
5-PI3K
6-LTB4
7-PPAR

41. Targeting protease activity at the
enzymatic level

42. Drugs that may have indirect anti-
inflammatory actions
Reversing glucocorticoid resistance :
Activation of HDAC2: theophylline;
curcumin; resveratrol
Inhibition of P-glycoprotein
Inhibition of MIF

43. These data are communicated for scientific purpose only. Confidential slide set
Bronchodilators in COPD

44. 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.

45. Bronchodilators
Indacterol Glycopyrronium bromide
Olodaterol Aclidinium bromide
Vilanterol
Xanthines

46. 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

47. 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.

48. 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;2014Lab.Clin

49. 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.

50. 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

51. 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

52. 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.

53. 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.

54. 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.

55. 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.