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This is to certify that the
dissertation entitled

CHARACTERIZATION OF DELTA-9-
TETRAHYDROCANNABINOL-MEDIATED
ALTERATIONS IN LEUKOCYTE AND PULMONARY
AIRWAY EPITHELIAL CELL RESPONSES TO A
PRIMARY CHALLENGE WITH INFLUENZA NPR/8/34
IN C57BL/6 WILD-TYPE AND CB1/CBZ RECEPTOR-
NULL MICE

presented by

JOHN PHILIP BUCHWEITZ

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Pharmacology and Toxicology

 

 

A

WWT

 

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3/2/ 0 7

Date

 

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6/07 p:/ClRC/DateDue.indd-p.1

CHARACTERIZATION OF DELTA-9-TETRAHYDROCANNABINOL-MEDIATED
ALTERATIONS IN LEUKOCYTE AND PULMONARY AIRWAY EPITHELIAL
CELL RESPONSES TO A PRIMARY CHALLENGE WITH INFLUENZA A/PR/8/34
IN C57BL/6 WILD-TYPE AND CB1/CB2 RECEPTOR-NULL MICE

By

John Philip Buchweitz

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Pharmacology and Toxicology

2007

ABSTRACT
CHARACTERIZATION OF DELTA-9-TETRAHYDROCANNABINOL-MEDIATED
ALTERATIONS IN LEUKOCYTE AND PULMONARY AIRWAY EPITHELIAL
CELL RESPONSES TO A PRIMARY CHALLENGE WITH INFLUENZA A/PR/8/34
IN C57BL/6 WILD-TYPE AND CBl/CBZ RECEPTOR-NULL MICE
By
John Philip Buchweitz
Delta-9-tetrahydrocannabinol (Ag-THC) is a well-established immunosuppressive agent
derived from the plant Cannabis Sativa. The biological activity of Ag-THC is presumed to
result from interactions of Ag-THC with the cannabinoid receptors CB; and C82. Ag-THC
has been demonstrated to effect innate, cell-mediated, and humoral immune responses.
Accordingly, A9-THC has been implicated as both a therapeutic agent toward
unwarranted humoral immune responses and as a determinant of susceptibility toward
infectious disease. Influenza is a common respiratory pathogen that affects millions of
people world-wide each year. Viral clearance from the respiratory tract occurs through a
combination of innate, cell-mediated, and humoral immune responses in the
immunocompetent host. Therefore, it was hypothesized that the intrinsic
immunosuppressive properties of Ag-THC could lead to decreased host resistance in mice
resulting in a greater viral burden when compared with uninfected mice. It was further
hypothesized that decreased host resistance to influenza would enhance morphologic
features consistent with cellular injury and repair along the bronchiolar epithelium lining
the airways of mouse lungs. Moreover, since clonal expansion of T cell populations
critical to viral clearance are dependent on IL-2, and IL-2 expression is modulated by

cannabinoids through a mechanism that is independent of CBl/CBZ receptors, the

proposed increased susceptibility to influenza in the current studies might also arise from

CB; and CB2 receptor-independent mechanisms. In the current studies, challenging mice
with influenza alone resulted in robust inflammatory responses that were comprised of a
diverse array of transient immune cells and their secretory chemical mediators in the
airways. In addition to the inflammatory response, there were concomitant alterations in
epithelial morphology that included cellular apoptosis and influenza virus-induced
mucous cell metaplasia (MCM). Treatment of influenza infected mice with Ag-THC led
to a dose-dependent increase in viral burden as compared to mice infected with influenza
alone. More importantly, Ag-THC treated mice exhibited decreased recruitment of critical
populations of CD4+ and CD8+ T cells and macrophages necessary for the clearance of
influenza as compared to mice infected with influenza alone. Lastly, there were
identifiable CB; and CB2 receptor-dependent and -independent mechanisms involved in
host immune responses to influenza infection. More specifically, the profile of viral
burden included an increase in H1 mRNA amongst mice treated with A9-THC that was
similar between CBf”/CB2'/' and wild type mice suggesting a CB; and CB2 receptor-
independent finding. Conversely, the magnitude of viral burden was strikingly less in
CBl'/’/CBz"‘ mice than in wild type mice which suggested the involvement of CB; and/or
CB; receptors in mediating immune homeostasis. In addition to viral burden, there were
other unique findings exhibited by CBl'/'/CB2'/' mice in response to Ag-THC treatment
alone as compared to Ag-THC treated wild type mice. Most notably, Ag-THC induced
MCM of the bronchiolar epithelium independent of PR8 challenge in CBl'/'/CBz'/' mice.
In conclusion, these findings suggest that Ag-THC is a determinant of susceptibility to

increased viral burden and that alternative receptors for Ag-THC are, in part, responsible.

This dissertation is dedicated to the men and women of our armed services who have

fought so bravely in the defense of my family’s freedoms. God Bless all of you.

iv

ACKNOWLEDGMENTS

First, I would like to extend my gratitude to my mentor, Norbert E. Kaminski,
whose guidance and encouragement has not gone unnoticed in my tireless pursuit of the
doctorate degree. In addition, I would like to thank my committee members, Dr. Alex
Chen, Dr. Jane Maddox, and Dr. Jack Harkema for their support and assistance.

My research was made all the more enjoyable by the camaraderie of my fellow
colleagues including Dr. Courtney Sulentic, Dr. Cheryl Rockwell, Dr. gautham Rao, Dr.
Jim Wagner, Dr. Aimen Farraj, Dr. Ammie Bachman, Dr. Barb Faubert Kaplan, Dr.
Steve Carey, Dina Schneider, Colin North, and Peer Karmaus. The research was also
made possible by the valuable assistance and skills of Lori Bramble, Bob Crawford,
Kimberly Hambleton and the histology group (Amy, Joselyn, Kathy, and Rick)... you
have all been a blessing.

Lastly, I would like to thank my family, Cynthia, Nathan, Olivia, for being

supportive and making each day more fulfilling.

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................................

LIST OF FIGURES .................................................................................................

LIST OF ABBREVIATIONS .................................................................................

INTRODUCTION ...................................................................................................

I.

II.

III.

Respiratory toxicology .................................................................................
A) Basic anatomy and physiology ...............................................................
B) Respiratory clearance of inhaled particles and pathogens ......................

1. Mucociliary clearance ......................................................................
2. Alveolar clearance ............................................................................

C) Epithelial cell death and regeneration ....................................................

1. Clara cells .........................................................................................
2. Type II cells .....................................................................................

Influenza Virus .............................................................................................
A) Classification ..........................................................................................
B) Viral Entry ..............................................................................................
C) Viral Clearance .......................................................................................
D) Chemokine and cytokine responses to influenza virus ...........................

E) Models of susceptibility ...........................................................................

Cannabinoids ................................................................................................
A) Chemistry of delta-(9)-tetrahydrocannabinol .........................................
B) Pharmacokinetics ....................................................................................

1. Absorption ........................................................................................
2. Distribution .......................................................................................
3. Metabolism .......................................................................................

C) Biological receptors and activity ............................................................

1. CB1 and CB2 receptors ....................................................................
2. Alternative receptors ........................................................................

D) Effects of cannabinoids on the immune system .....................................

13)

1. Effects on macrophage function .......................................................
2. Effects on neutrophil function ..........................................................
3. Effects on natural killer cell function ...............................................
4. Effects on THI cells ..........................................................................
5. Effects on CD8+ T cells ....................................................................
6. Effects on immune cell-derived chemokines and cytokines ..............
Models of host-resistance .......................................................................
1. Model of A9-THC and Listeria monocytogenes ...............................

vi

X

xi

xiv

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p—i

15
15
15
16
16
18
18
18
19
2O
20
21
22
23
24
24
25
25

2. Model of Ag-THC and Herpes Simplex Virus .................................

3. Model of Ag-THC and Legionella pneumophila ..............................

IV. Objectives .....................................................................................................

MATERIALS AND METHODS ............................................................................
I. Cannabinoid compounds ......................................................................
11. Animals ................................................................................................
III. Experimental designs ...........................................................................
IV. Influenza A/PR/8/34 instillation ..........................................................
V. Steady state blood serum levels of Ag-THC and its metabolites ...........
VI. Necropsy, lavage collection, and tissue preparation .............................
VII. Immunocytochemistry .........................................................................
VIII. Total and PCNA positve epithelial numeric cell density and labeling
index ........................................................................................................
IX. CAS-3 and alcian blue numeric cell densities ......................................
X. Morphometry of stored intraepithelial mucosubstances ......................
XI. Bronchoalveolar lavage cellularity ......................................................
XII. Histopathology scores for inflammation ..............................................
XIII. Total protein .........................................................................................
XIV. Neutrophil elastase ...............................................................................
XV. Inflammatory and THI/TH2 cytokines ................................................
XVI. T-lymphocyte flow cytometry ..............................................................
XVII. RNA isolation ......................................................................................
XVIII. Statistical Analysis ...............................................................................
EXPERIMENTAL RESULTS ..............................................................
1. Model developmentzEstablishing the dose ...................................................

II.

A) Concentration-dependent inflammatory responses to PR8 in pulmonary
airways ....................................................................................................
B) Steady state blood serum levels of A9-THC and its metabolites ............

Time-dependent airway epithelial and inflammatory cell responses
induced by influenza virus A/PR/8/34 in C57BL/6 mice .............................
A) PR8 induces time-dependent alterations in epithelial morphology ..........
B) Temporal analysis of stored intraepithelial mucosubstances following
PR8 challenge ......................................................................
C) Expression levels of whole lung MUCSAC mRNA at 7 days post
Infection
D) Time-dependent differences in total and PCNA immunopositive
epithelial numeric cell densrties
E) Bronchoalveolar lavage fluid analysis ....................................................

vii

26
27

29

30

30
3O
30
31
31
31
34

34
34
35
36
36
37
37
38
39
39
40

41

41

41

41

45

45

49

49

49
54

1. Protein ............................................................................................... 54

2. Total and differential inflammatory cell counts ................................ 54

3. inflammatory chemokines and cytokines .......................................... 54

4. TH2 cytokines IL-4, IL-5, IL-9 and IL-13 ........................................ 58

5. Elastase .............................................................................................. 58

III. Modulation of airway responses to influenza A/PR/8/34 by A9-

tetrahydrocannabinol in C57BL/6 mice ......................................................... 62
A) Ag-THC increases whole lung H1 mRNA following PR8

challenge... 62
B) A 9-THC does not affect total protein levels in BALFM ........................... 62
C) A 9-THC decreases leukocyte populations retrieved in BALF ................ 62
D) Ag-THC decreases CD4+ and CD8+ T cells in BALF ............................ 66
E) Ag-THC modestly affects chemokines and cytokines in retrieved in

BALF ...................................................................................................... 66
F) A9—THC treatment reduces inflammation scores for mouse lung

sections ............................................................................ 70
G) Effect of A9-THC on the observed pulmonary histopathology to PR8.... 70
H) Caspase-3 mRNA expression levels and G5 numeric cell densities ....... 75
I) MUCSAC mRNA expression levels and G5 numeric cell densities ....... 76
J) A9-THC modestly enhances neutrophil-derived elastase levels in

IV. Pulmonary airway responses to influenza A/PR/8/34 in CB1'/'/CB;'/' mice
exposed to A9-tetrahydrocannabinol: An examination for the role of

cannabinoid receptors .................................................................................... 81
A) Qualitative health assessment ................................................................. 81
B) The viral load of PR8 in the pulmonary airways of CB.'/'/CB;' ' and
wild type mice measured 7 days after challenge ............................. 83
C) A 9-THC affects vascular permeability induced by Pr8 infection of the
airways in CB] "/CB;' and wild 9type mice ............................................. 83

D) CB1 / "/CB;' mice treated with A 9-THC exhibit a distinctly different
composition of leukocytes recruited to the airways in response to PR8

infection when compared to wild type mice. . . . . . .. ................................. 85
E) BALF-associated CD4+ and CD8+ T cell levels following PR8
challenge in CB|'/'/CB;'/° and wild type mice .......................................... 90

F) Cannabinoid receptor deficient mice exhibit unique differences in
epithelial and leukocytic Chemokine and cytokine secretion in the

pulmonary airways... 93
G) Cytokines and chemokines detected 1n blood serum .............................. 98
H) The absence of cannabinoid receptors CB. and CB; enhances the

observed pulmonary histopathology ....................................................... 101
I) A9-THC affects the magnitude of the inflammatory response to PR8 1n

wild type and CB; and CB; deficient mice .............................................. 104
J) Effects of C81 and CB; deficiency on the numeric cell densities of

apoptotic cells and metaplastic goblet cells ............................................. 104

viii

DISCUSSION .......................................................................................................... 109

1. Animals utilized in these studies ................................................................ 109
II. The method of delivery and the dose of PR8 utilized .. ............................. 109
III. The method of delivery and dose of A9-THC utilized in these studies ..... 109
IV. Effect of A9-THC on viral H1 mRNA levels in wild type and CBI'I'

/CB;'/' mice ..................................................................................................... 1 l 1

V. Effect of PR8 on inflammatory cell recruitment to the pulmonary
airways in the presence or absence of A9-THC in wild type and CBl'/'
/CB;'/'mice ..................................................................................................... l 13
VI. Effect of PR8 on the release of soluble mediators into the pulmonary
airways in the presence or absence of A9-THC in wild type and CBI'I'

/CB;"'mice ..................................................................................................... 1 15
VII. Effect of a single PR8 instillation on pulmonary histopathology in the
presence or absence of Ag-THC in wild type and CB1'/'/CB;"' mice ............ 119

VIII. Effect of a single PR8 instillation on epithelial cell apoptosis and MCM
in the presence or absence of A9-THC in wild type and CB1'/'/CB;'/' mice.. 121

IX. Significance and relevance ....................................................................... 123
X.
LITERATURE CITED ............................................................................................. 128
APPENDIX .............................................................................................................. 140

ix

LIST OF TABLES

. Analysis of A9-THC, 9-COOH-A9-THC, and ll-OH- A9-THC
concentrations in mouse blood serum ............................................................ 44

. Total and differential cell counts from BALF 67

. CD4+ and CD8+ T cell populations observed in BALF by flow
cytometry ................................................................................. 68

. Inflammatory cytokines quantified in BALF by cytometric bead
array ....................................................................................... 69

. Summary of the effects of corn oil and A9-THC on BALF and

histochemistry measurements taken for epithelial and inflammatory cells
in wild type and CB;"’/CB;'/' mice ................................................ 110

10.

11.

12.

13.

14.

15.

16.

LIST OF FIGURES

Diagram of human (A) and mouse (B) lungs ...........................
Structure of A9-tetrahydrocannabinol ....................................

Diagram of the microdissection of mouse right lung lobes (R1, R2,
R3, R4) and left lung lobe at generations 5 (G5) and 11 (G11) ......

Increasing concentrations of influenza virus result in increasing
inflammatory responses in the hilar region of the left lung lobe at
generation 5 of the main axial airway ....................................

Bronchiolar epithelial morphometry following influenza infection.

Time-to-onset of mucous cell metaplasia following influenza
infection ......................................................................

MU C5AC gene induction by influenza at 7 days post infection. . .

Time-dependent proliferation of epithelial cells lining the main
axial airway following influenza infection ..............................

Time-dependent detection of total protein in bronchoalveolar
lavage fluid following influenza infection ...............................

Time-dependent recruitment of leukocytes to the pulmonary
airways following influenza infection ....................................

Secretion of chemokines and cytokines into the airways in
response to influenza infection ............................................

Detection of TH2 cytokines secreted into the airways in response to
influenza infection ..........................................................

Secretion of neutrophil-derived elastase into the airways in
response to influenza infection ............................................

A9-THC treatment enhances hemagluttinin 1 mRN A levels ..........
A9-THC does not alter total protein levels in BALF ...................

A9-THC decreases inflammation scores in a time-dependent
manner ........................................................................

xi

17

33

43

47

50

51

53

55

57

60

61

63

64

65

72

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

Examination of the effects of Ag-THC on the inflammatory
response to influenza challenge by histopathology .................... 74

A9-THC has no effect on Caspase-3 mRNA levels and epithelial
numeric cell densities ...................................................... 78

A9-THC increases MUCSAC mRNA levels and decreases
epithelial numeric cell densities for acidic mucosubstances .......... 80

A9-THC modestly increases neutrophil-derived elastase levels
secreted in the pulmonary airways ....................................... 82

Effects of A9-THC treatment on the expression levels of H1 mRNA
in CB.""/CB;'/' and wild type mice ....................................... 84

Effects of A9-THC treatment on total protein in BALF in CB1" ’
/CBz"' and wild type mice ................................................. 86

Effects of A9-THC treatment on total and differential leukocyte
counts in BALF in CB1'/'/CB;‘/' and wild type mice ......................... 89

Effects of A9-THC treatment on CD4)r and CD8+ T cells retrieved
in BALF in CB1'/'/CB;"' and wild type mice ............................ 92

Effects of A9-THC treatment on inflammatory chemokines and
cytokines in secreted into the pulmonary airways in CB1'/'/CB;'/'
and wild type mice .......................................................... 95

Effects of Ag-THC treatment on TH2 cytokines secreted into the
pulmonary airways in CBt'/'/CB;'/' and wild type mice ................ 97

Effects of A9-THC treatment on inflammatory chemokines and
cytokines released into blood serum in CBl'/'/CB;'/' and wild type
mice ........................................................................... 100

Inflammatory res onse to PR8 in the proximal section of the left
lung lobe in CB1' '/CB;'/' and wild type mice ........................... 103

Effects of A9-THC treatment on histopathology-based
inflammation scores in CB1’/'/CB;'/' and wild type mice ............. 105

Effects of Aq-THC treatment on the numeric cell density of
caspase-3 positive epithelial cells in CB,'/'/CB;"' and wild type

mice ........................................................................... 1 06

xii

31. Effects of A9-THC treatment on the numeric cell density of
mucosubstances in airway epithelial cells in CB1'/’/CB;'/' and wild
type mice ..................................................................... 108

xiii

AB/H
AB/PAS
ABS
AIDS
ANOVA
AP-l
B-l

13-2
BALF
BCA
CAS-3
CBl
CB1"
CB;

CB;'/'

CBi"'/CB;"’

CD
cDNA
C/EBPB
CNS
CO

CTL

LIST OF ABBREVIATIONS
Alcian blue/hematoxylin
Alcian blue/periodic acid schiff
Automation buffer solution
Acquired Immune Deficiency Syndrome
Analysis of variance
Activating protein-1
Non-antigen stimulated B cell
Antigen stimulated B cell
Bronchoalveolar lavage fluid
Bicinchoninic acid
Caspase-3
Cannabinoid receptor type 1
Cannabinoid receptor type 1 null
Cannabinoid receptor type 2
Cannabinoid receptor type 2 null
Cannabinoid receptors type 1 and 2 null
Cluster of differentiation
copy-deoxynucleic acid
Nuclear factor-IL-6
Central nervous system
Corn oil

Cytotoxic T lymphocytes

xiv

AKTHC
DNA
dpi

F ACS

F asL
FCM
GS

G11

H&E
HSVl
HSV2
icv
IFN or/[i
IFN-y
IL
IP-10
IRF
KO
LAK
LD50
M1

MCM

Delta-9-tetrahydrocannabinol
Deoxynucleic acid

Days post-infection

Flourescence assisted cell sorting
F as ligand

Flow cytometer

Airway generation 5

Airway generation 11
Hemagglutinin

Hematoxylin and eosin

Herpes simplex virus type 1
Herpes simplex virus type 2
intracerebroventricularly
Interferons -alpha and -beta
Interferon-gamma

Interleukin
Interferon-gamma—inducible protein
Interferon regulatory factors
Knockout

Lymphocyte activated killer cells
Lethal dose to 50 percent of the population
Matrix protein 1

Mucous cell metaplasia

XV

MCP-l
MCP-3
MDCK
MHC
MIP 1 -0l
MIP 1 -|3
MIP-2
MIP3-a

mRNA

NFAT
NF -1<B
NIH
NK
NP
NS
PA
PB 1
PB2
PAS
PCNA
pfu

PMA

Monocyte chemoattractant protein-1
Monocyte chemoattractant protein-3
Madin-Darby Canine Kidney cell

Major histocompatibility complex
Macrophage inflammatory protein l-alpha
Macrophage inflammatory protein l-beta
Macrophage inflammatory protein 2
Macrophage inflammatory protein 3-alpha
Messenger ribonucleic acid
neuraminidase

Nuclear factor of activated T cells
Nuclear factor kappa B

National Institutes of Health

Natural killer cells

nucleoprotein

nonstructural protein

Polymerase type A

Polymerase type B1

Polymerase type 82

Periodic acid schiff

Proliferating cell nuclear antigen

Plaque forming units

Phorbol myristate acetate

xvi

PMN

PR8

RANTES

RT-PCR

SAL

SEM

STAT

Polymorphonuclear leukocyte

Influenza A/PR/ 8/34

Regulated on activation, normal T expressed and secreted
Ribonucleic acid

Reverse transcriptase-polymerase chain reaction
Sahne

Standard error of the mean

Signal transducers and activators of transcription
T-helper 1 lymphocyte

T-helper 2 lymphocyte

Tumor necrosis factor-alpha

Transforming growth factor-beta

Volume density

xvii

INTRODUCTION

1. Respiratory toxicology

The primary function of the respiratory system is the exchange of gases (e.g.,
oxygen and carbon dioxide) across the alveolar and capillary beds. Gas exchange is so
important to the function of all other systems in the body that merely minutes without
oxygen will lead to the death of the host. Therefore, noxious gases, particulates and
pathogens that perturb normal lung function have life threatening potential. To better
understand the responses of the lung to toxicant or pathogen insult, a basic background

on the anatomy and physiology of the respiratory tract is provided.

A) Basic anatomy and physiology

The respiratory tract can be divided into two parts: 1) the upper respiratory tract,
and 2) the lower respiratory tract. The upper respiratory tract consists of the nasal
airways, larynx, and trachea. While the lower airways consist of the trachea and right and
left lung lobes which contain bronchi, bronchioles, terminal bronchioles, alveolar ducts
and alveoli.

In the nasal cavity of the upper respiratory tract, incoming air is filtered, for the
removal of large particulate matter, and conditioned (humidified) for passage into the
lower respiratory tract. In addition, chemical odors are processed by the olfactory
epithelium and olfactory sensory neurons to provide a sense of smell. From a
comparative standpoint, humans breathe nasally and orally (through the mouth), whereas
mice are obligatory nose breathers. Developmentally, the nasal cavity in man is relatively

simple in comparison to that of the rodent. Increased specialization in the nasal cavity of

mice that includes increased numbers of olfactory epithelial cells which emphasizes a
primary function of the nose for olfaction in mice (1).

From the upper respiratory tract, the trachea bifurcates into the right and left lung
lobes. In humans, the left lung lobe is divided by an interlobular fissure into two lobes
(superior and inferior), and the right lung lobe is also divided by interlobular fissures into
three lobes (superior, middle and inferior) (Figure 1A). Comparatively, the mouse right
lung is divided into four structurally distinct lobes (apical, middle, caudal, and
accessory), and the mouse has a single left lung lobe (Figure 1B). Structurally, the lower
respiratory tract can be further divided into the conducting airways and the respiratory
airways. The conducting airways consist of the trachea, bronchus, secondary and tertiary
bronchi and non-respiratory bronchioles. The respiratory bronchioles consist of alveolar
ducts and alveoli.

The epithelium lining the larger conducting airways is predominantly
pseudostratified and columnar whereas smaller conducting airways are primarily
comprised of cuboidal epithelial cells. The cellular makeup includes clara cells, basal
cells, ciliated epithelial cells and mucin-producing, goblet cells. Together, these cells
provide an important defense mechanism for the lung, termed the mucocilliary escalator.
The mucociliary escalator is regulated by the production of mucous by goblet cells and
the beating of cilia. The movement of cilia is under the control of the autonomic nervous
system.

The alveoli are composed of type I and type 11 cells. The Type I cell is a thin cell
stretched over a very large area. Type 1 cells cannot replicate and are susceptible to a

large number of toxic insults. Type I cells are responsible for gas exchanges occurring in

 

Figure 1. Diagram of human (A) and mouse (B) lungs. (Not drawn to scale).

the alveoli. The Type 11 cell is a smaller, roughly cuboidal cell that is usually found at the
alveolar septal junction. Type 11 cells are spherical cells which comprise only 4% of the
alveolar surface area, yet they constitute 60% of alveolar epithelial cells and 10-15% of
all lung cells. Four major functions have been attributed to alveolar type 11 cells: 1)
synthesis and secretion of surfactant; 2) xenobiotic metabolism; 3) transepithelial
movement of water; and 4) regeneration of the alveolar epithelium following lung injury

(2, 3).

B) Respiratory clearance of inhaled particles and pathogens

A variety of different particles, such as bacterial or viral agents and environmental
pollutants are deposited in the lungs while breathing. Three main clearance mechanisms
operate in the lungs of humans to remove the inhaled and deposited materials as well as
cellular debris in order to keep the airways relatively clean. Mucociliary clearance clears
the conducting airways of its own secreted mucus, together with substances trapped in it,
by means of the mucociliary escalator. Coughing serves as a backup system if
mucociliary clearance fails. Lastly, alveolar clearance removes insoluble particles
deposited on the respiratory surface of the lungs (4). In contrast to humans, mice do not

possess a cough reflex.

1) Mucociliary clearance
Mucus behaves as a viscoelastic gel, consisting of water and high molecular mass
cross-linked glycoproteins mixed with serum and cellular protein, including albumin,

enzymes, immunoglobulins, and lipids (4). Ciliary movement in the conducting airways

propels mucus in a cephalic direction to be swallowed or expectorated. If levels of mucus
exceed the threshold by which normal ciliary movement cannot propel the mucus in a

cephalic direction, then the potential for mucus plugging exists (5).

2) Alveolar clearance.

Small particles from 2 to 0.2 microns in diameter can escape the defense
mechanisms of the upper respiratory tract and the mucociliary escalator to reach the
alveoli. Pathogens such as bacteria and viruses are generally less than 2 microns in
diameter. Once these microorganisms arrive in the alveoli they are handled in different
ways.

Bacteria can be opsonized by immunoglobulins present in the fluid lining the
alveoli. The opsonized bacterium is readily phagocytosed by resident alveolar
macrophages. If the organism bypasses opsonization then the macrophage may still be
able to phagocytose the bacterium, however, at a much slower rate. Once the
microorganism is phagocytosed by the macrophage, the organism is destroyed within an
endosome and small microbial protein fragments are presented on the surface of the
macrophage in the context of type 11 major histocompatability complexes (MHC II) to
awaiting T cells. Once activated, T cells can signal B cells to produce more antibody
and/or activate macrophages to increase phagocytic activity and release soluble chemical
mediators that help recruit polymorphonuclear neutrophils (PMN) from the blood stream.
PMNs are also phagocytic cells that work optimally in conditions where antibody and
complement are present.

Viruses, unlike bacteria, are obligate intracellular parasites that replicate inside

the epithelial cells lining the airways of the lung. Viral clearance is accomplished by cell-
mediated immunity. Cell-mediated immunity involves the activation of T cell subsets into
effector T cell populations that include cytotoxic T cells, and T helper cells of the THl
and TH2 subtypes. During a cell-mediated immune response to viral infection, antigens
are presented to CD8Jr T cells in the context of a major histocompatability complex I
(MHC I), that is present on all nucleated cells. Conversely, CD4+ THI and TH2 cells
detect antigens in the context of MHC 11 presented by macrophages, dendritic cells and B
cells. Once CD8+ T cells recognize host cells bearing new antigens in the context of
MHC I, the cytotoxic T cell releases cytolytic proteins (perforin) that form holes in the
membrane through which granzyme is delivered to induce apoptosis of the target cell.
The process is continued until all infected cells have been cleared. There is growing
support for the role of CD4+ T cells as contributing immune effectors in the protection
against influenza (6-8). CD4+ T cells carry out a supportive role through the promotion of
long-lasting CD8+ T memory cells, mediating the clearance of virus by an interferon-
gamma (IFN-y)-dependent mechanism, or through direct cytolytic effects on infected
cells, or by a combination of these functions (7). The direct cytolytic activity of CD4+
cytolytic T cells (CTL's) through Fas-Fas ligand (Fas-FasL) interactions has been
previously established (9, 10). These findings illustrate the redundancies found within the

acquired immune response that collectively defend the host against infectious pathogens.

C) Epithelial cell death and regeneration.
The cells of the lung are under constant insult by airborne chemicals or pathogens.

Typically these insults are dealt with by the inherent functional properties of the lung

(e.g. mucociliary escalator, resident alveolar macrophages, and the cough reflex).
However, when chemical agents or viral pathogens target cells directly for death,
mechanisms to replace the dying cells must be in place. Specialized cells like the Clara

cell and the type 11 cell are responsible for replicating and differentiating to fill the void.

1) Clara cells

In the conducting airways, Clara cells with enhanced epidermal growth factor
receptor expression can be activated by multiple stimuli to differentiate into goblet cells.
Differentiating into increased numbers of goblet cells, also known as mucous cell
metaplasia, (MCM) has the potential to lead to a state of hypersecretion of mucus that
contributes to airway plugging.

MCM has been well characterized in rodent models that exhibit airway
remodeling after allergen exposure (11-13) as well as after toxicant exposure (13-15).
The pathogenesis of MCM in these models appears to be intimately linked with soluble
mediators derived from pro-inflammatory neutrophils and macrophages as well as TH2
cells during the acquired immune response. For instance, neutrophil-derived elastase
(16), tumor necrosis factor — alpha (TNF-a) (17) and the TH2 cytokines IL-4 (18), IL-5
(19), IL-9 (20), and IL-13 (21) have been implicated in the etiology of MCM. More
recently, there has been an increasing body of evidence that MCM also occurs as a
component of the progressive changes in respiratory pathology in models of viral
infections (22) including adenovirus (23), respiratory syncytial virus (RSV) (24) and

influenza virus (25).

2) Type 11 cells.
In the alveoli, type 11 cells replace dying type I and type 11 cells. The type 11 cell is
a pluripotent cell that can divide and differentiate into type I cells. Efforts to further
characterize type 11 cell proliferation and differentiation in vitro utilizing freshly isolated
type 11 cells, however, has been met with difficulty. In particular, the isolation procedure

has been suggested to result in the blockage of cell cycling as a cell stress response (26).

II. Influenza Virus

A) Classification

Influenza viruses are enveloped, negative-stranded RNA viruses belonging to the
family of Orthomyxoviridae. Influenza A virus RNA is composed of eight segmented
genes, which encode for ten different proteins (27). These proteins include: the envelope
glycoproteins hemagglutinin (H) and neuraminidase (N), matrix protein (M1),
nucleoprotein (NP), three polymerases (PBl, PB2 and PA), ion channel protein M2, and
nonstructural proteins N31 and NS2. Influenza A viruses are classified according to their
H (HI—H15) and N (N l—N9) genes. Viruses with hemagglutinin types H1, H2 and H3
and neuraminidase types N1 and N2 are pathogenic in humans. The nomenclature of
influenza viruses provides detailed information regarding virus type and origin. For
example, the nomenclature for influenza virus A/PR/8/34 (HlNl) indicates that the virus
type is influenza virus type A, originating from Puerto Rico, as the 8th isolated strain, in
the year 193_4. Additionally, the surface proteins H and N subtype the virus as an

influenza virus that belongs to the group of influenza viruses designated HlNl.

B) Viral Entry

Influenza viruses preferentially replicate in the epithelial cell layers of the upper
respiratory tract; however, macrophages and other leukocytes may also be infected (28).
Most cell types carrying the influenza A virus receptor, sialic acid containing cell surface
glycoproteins, are susceptible to the virus (29). Recent work by Chu and Whittaker
(2004) (30) has provided evidence that a cell line deficient in terminal N-linked
glycosylation, caused by a mutation in the N-acetylglucosaminytransferase I gene, could
not be infected by influenza virus. The cells were not deficient for total cell-surface sialic
acid and, accordingly, were able to bind the virus. However, viral binding to sialic acid
was not sufficient for infection, suggesting that virus binding to the sialic acid moiety
merely brought the virus into close proximity of a secondary receptor that triggered

internalization of the virus.

C) Viral Clearance

Myeloid cells such as neutrophils and macrophages, and acquired immune cells
like cytolytic and helper T cells mediate the clearance of virally infected epithelial cells
within the pulmonary airways. In addition, B cells provide support against subsequent re-
infection through the secretion of antigen-specific antibodies.

The contn'butions of phagocytic cells like the neutrophil and macrophage to the
complex process of viral clearance have been previously studied. Tsuru and coworkers
(1987) (31) examined phagocyte-depleted mice challenged intravenously with a sublethal
inoculate of influenza virus. Phagocyte depletion was accomplished by either

carrageenan treatment or gamma-irradiation. Neutrophils are sensitive to gamma-

irradiation and resistant to carrageenan treatment, whereas macrophages and natural
killer cells are gamma-insensitive and carrageenan-sensitive. Mice treated by gamma-
irradiation and challenged with influenza exhibited increased viral titres and death by 8
days. Conversely, mice treated with carrageenan cleared the virus by day 7. In addition,
the adoptive transfer of syngeneic neutrophils into gamma-irradiated mice was not
accompanied by increased viral titres. Hence, the neutrophil, and not the macrophage,
was suggested to play an important role in early phase responses to influenza.

CD8+ T cells are cytolytic T cells that induce the apoptotic cell death of virus-
infected cells upon antigen recognition and coordinated co-stimulation. Allan et al.,
(1990) (32) demonstrated that CD8+ T cells mediated the clearance of influenza virus
from the lung. Specifically, the authors depleted mice of CD4+ T cells and subsequently
challenged mice with influenza virus A (H3N2). An increase in the mediastinal lymph
node size during influenza infection was not observed with CD4+ T cell depletion. In
addition, elimination of CD4+ T cells did not influence the severity of the inflammatory
response in the lung. Moreover, mice deficient of CD4+ T cells were capable of clearing
the virus infection, suggesting that CD8+ T cells were the most important of the two T
cell types for developing an effective cell-mediated immune response against influenza.

Although CD4+ T cells were not required for viral clearance, the role for CD4+ T
cells in the immune response to influenza was unclear. Graham et al., (1994) (33)
examined the contribution of CD4+ T cell subsets. Influenza virus-specific THI and TH2
clones were generated from influenza-primed BALB/c mice. THI clones were shown to
have cytolytic properties in vitro and were protective against a lethal challenge of virus in

vivo. TH2 clones were nonprotective and mice exhibited an exacerbation of the

10

pulmonary pathology associated with influenza infection. The results suggested that THl
cells provided support against primary influenza infections. Likely, THI cells through the
release of IFN-y elicited a cell-mediated immune response against influenza-infected
cells.

Moran et a1 (1999) (34) recognized that immunization with live virus expanded
TH] memory cells and provided protection against a heterosubtypic (different viral
surface marker subtype) viral challenge and that immunization with inactivated virus
expanded TH2 memory cells without affording the host protection. Hence, the authors
created a THl priming environment with the inclusion of IL-12 and antibodies to IL-4
along with the inactivated virus to yield a TH] memory cell population that facilitated the
generation of cytotoxic T cells upon challenge with a heterosubtypic virus.

Antibodies play an important role in targeting specific viral surface proteins like
hemagglutinin and neuraminadase to neutralize and more importantly opsonize the virus
for clearance by phagocytic cells. Therefore, the humoral immune response is beneficial
to the host with subsequent re-infection. Baumgarth et al., (2000) (35), studied the
contribution of B cells and secretory immunoglobulin M in protection against influenza
virus. The authors found that 129/Sv mice deficient in secretory IgM but capable of
expressing surface IgM had reduced viral clearance and survival rates. More importantly,
both B-l (non-antigen stimulated) and B-2 (antigen stimulated) cell-derived IgM
antibodies were deemed necessary for the survival of the host. These findings support the

role of antibodies in neutralizing and opsonizing viruses for clearance by phagocytes.

D) Chemokine and cytokine responses to influenza virus

11

The antiviral state created by influenza infection of the respiratory epithelium and
alveolar macrophages of the lung results in the production of chemotactic [regulated on
activation, normal T expressed and secreted (RANTES), macrophage inflammatory
protein l-alpha (MIP-10L), monocyte chemoattractant protein-1 (MCP-l), monocyte
chemoattractant protein-3 (MCP-3), and interferon-gamma-inducible protein-10 (IP-10)],
pro-inflammatory (IL-H3, IL-6, IL-18, and TNF-a) and anti-viral [interferons -alpha and
-beta (IFN-on/Bfl cytokines (36). The production of specific chemokines and cytokines
provide a feedback loop that supports THI cell-mediated immunity against influenza-
infected epithelium.

Chemokines are produced by a variety of cells constitutively or in response to
microbial infections. Chemokines bind to their specific cell surface receptors on
leukocytes, enabling them to migrate from blood vessels through the vascular
endothelium into the site of inflammation (37). Influenza virus infected macrophages
secrete MIP-1a, MIP-1b, RANTES, MCP-l, MCP-3, MIP-3a and IP-10 (38-40) whereas
epithelial cells produce RANTES, MCP-l and IL-8 (41, 42). Antiviral (IFN-ol/B) and
immune-stimulatory cytokines (IL-113, lL-6, IL-18, and TNF-a) are largely produced by
influenza-infected macrophages (28, 43-45) and to a lesser extent by influenza infected
epithelial cells (28, 43, 46). Dendritic cells are the primary source of IL-12 production
(47). IFN-a/B is important in the recruitment of monocytes/macrophages and THl cells
through the upregulation of MCP-l, MCP-3, and IP-lO (37, 48). In addition, IFN-
a/B enhances antigen presentation by upregulating MHC gene expression (49).
Moreover, IFN-a/B is involved in T cell survival (50) and enhancement of IFN-y

production in natural killer cells (NK) and T cells. Conversely, the pro-inflammatory

12

cytokines IL-IB, IL-6, and TNF-a do not directly contribute to the anti-viral activity of
leukocytes (49). IL-lB and TNF-a enhance MCP-l gene expression and maturation of
tissue macrophages. NK and T cell derived IFN-y primes macrophages for increased
cytokine production, thereby leading to a positive feedback loop between macrophages
and NK and T cells (36).

The induction of chemokines and cytokines by influenza infection involves the
activation of transcription factors. Nuclear factor kappa B (NF-KB), activating protein
(AP-1), interferon regulatory factors (IRFS), signal transducers and activators of
transcription (STATS) and nuclear factor-IL-6 (C/EBPB) have all been Shown to be
activated during influenza infection (40, 46) (51-54). NF-KB activation occurs during
viral replication and protein synthesis (46). Virus replication has also been shown to
activate the transcription factor AP-l (54). The activation of STAT, on the other hand, is

indirectly mediated by virus-induced IFN-a/B (40, 46).

E) Models of susceptibility

Increased susceptibility to influenza virus infection following exposure to
respirable chemicals or particulates has been a human health concern for people whose
occupations include increased risk to inhaled particulate matter. In particular, research
has been conducted to address the concern with environmental chemicals and particulates
such as coal particulates, diesel exhaust, and ozone.

Coal miners working underground are chronically exposed to respirable
particulates of coal and emissions from diesel engines. Studies have been conducted in

mice to determine whether coal miners are at increased risk for developing respiratory

l3

infections like influenza virus. Hahon et a1. (1985) (55) examined the susceptibility to
influenza infection in CD-l white Swiss mice exposed to coal dust, diesel emissions, or a
combination of both for 1, 3, and 6 months. The course of influenza infection in mice did
not differ among treatment groups with respect to mortality and duration of exposure to
particulates. However, there was an increased severity of reaction to influenza in mice
treated with diesel emissions when compared to filtered air controls and mice receiving
coal dust.

To elucidate why the severity of the response to influenza was greater with
exposure to diesel emissions, Jaspers et al., (2005) (56) utilized an in vitro model of
human respiratory epithelial cells to determine the effects of aqueous trapped diesel
exhaust on influenza A/Bangkok/1/79 infection. Exposure of the respiratory epithelial
cells to diesel exhaust resulted in increased influenza infection. Increased infection,
however, was not attributed to the suppression of antiviral mediators. Alternatively, the
authors suggested that increased oxidative stress generated during exposure to diesel
exhaust enhanced viral attachment and infection of the epithelial cells. The role of
oxidative stress has been supported by more recent work in which the addition of
glutathione in vitro and in vivo resulted in decreased viral titres with increasing
concentrations of glutathione and an inhibition of influenza-induced activation of
apoptotic caspases and F as as compared to controls (57).

Human exposure to ozone, the primary oxidant gas in photochemical smog, has
been associated with altered pulmonary function and airway reactivity (58) and airway
inflammation (59). Therefore, exposure to ozone might also render individuals more

susceptible to respiratory infections. Wolcott et al., (1982) (60) exposed Swiss-Webster

14

mice to ambient levels (0.5ppm) of ozone during an infection with influenza virus (WSN
strain) and demonstrated that infected mice exposed to ozone exhibited a reduced severity
of disease, as measured by a decrease in the mortality and delayed time to death. In
addition, the infected mice exposed to ozone exhibited differential distribution of the
virus with respect to filtered air controls. In particular, mice that were exposed to ozone
had no airway epithelial viral-antigen staining; rather the viral-antigen staining was
distributed to focal regions of infection in alveolar fields. In contrast, the virus infected
the respiratory epithelium in air-filtered controls and dispersed with time to the alveolar
parenchyma with relatively even distribution. Since ozone exposure has been Shown to
induce extensive desquamation of the respiratory epithelium and result in a regenerative
epithelium that consists of cells that morphologically represent squamous epithelilun (61-
63), the pulmonary changes observed might result from an airway lining that is more

resistant to infection by influenza virus (60).

III. Cannabinoids

A) Chemistry of delta-(9)-tetrahydrocannabinol

Marijuana, a crude preparation of Cannabis sativa, iS comprised of over 426
chemical entities. More than 60 of these compounds are known collectively as
cannabinoids (64). Mechoulam and Gaoni first described the isolation of cannabinoid
compounds from marijuana extracts in 1965 (65). Turk and coworkers (1971) (66) were

the first to isolate and identify delta-9-tetrahydrocannabinol (Ag-THC) (Figure 2).

B) Pharmacokinetics

15

1) Absorption

Cannabinoids are more readily absorbed following inhalation as compared to oral
consumption. Moreover, it has been shown that the vehicle in which A9-THC is
administered has an influence on the pharmacokinetics and, hence the effectivity of the
drug (67). For example, in a study conducted by Coper et a1, (l971)(68) utilizing the
same dose of Ag-THC administered in either polyethylene glycol or rape oil (canola oil),
rats exhibited decreased body temperature with A9-THC in polyethylene glycol and no
change in body temperature with A9-THC in rape oil. Likewise, a study conducted by
Perez-Reyes et al., (1973) (69) in humans demonstrated that radiolabeled 3H-Ag-THC,
when administered orally, was absorbed at different rates depending on the vehicle
utilized. However, there were no apparent differences in the overall psychotropic effects,

metabolism or excretion of the drug.

2) Distribution

The distribution of cannabinoids and their metabolites occurs based on the
physical and chemical properties of the drug. Cannabinoids are highly lipophilic
compounds. Therefore, their distribution among tissues, once they are absorbed, is
dependent on blood circulation and the hepatic and extrahepatic metabolism of the parent
compound.

Klausner and Dingell (1971) (70) quantified the tissue distribution of
intravenously administered 14C-Ag-THC in rats. The highest concentration of
radioactivity was measured in the lung and the lowest concentration in the brain. Further

studies conducted by Ho et al., (1970) (71) utilized 3H-Ag-THC administered by

16

Figure 2. Structure of A9-tetrahydrocannabinol

l7

inhalation. The tissue distribution was Similar to the findings by Klausner and Dingell.

3) Metabolism
The metabolism of Ag-THC results in a number of metabolites that include 11-
hydroxy-Ag-THC, 9-carboxy-A9-THC, and 8,11—dihidroxy-A9-THC. Since human
exposure to A9-THC commonly occurs via inhalation or oral ingestion, likely Sites of
metabolism might be the alveolar type 11 cells of the lung, the microflora of the gut, and

hepatocytes of the liver.

C) Biological receptors and activity
1) C81 and CB2 receptors

Cannabinoid compounds produce a myriad of biological effects in animals and
humans including effects on the central nervous system (CNS), cardiovascular,
endocrine, immune, and reproductive systems (64). The most widely studied of these are
the CNS and immune systems.

The putative mechanism by which cannabinoids have been proposed to mediate
their biological effects, at least in part, is through the cannabinoid receptors (CB1 and
CB;). The CB1 receptor (brain receptor) was first cloned and characterized for expression
by Matsuda and coworkers (72). Likewise, the CB; receptor (peripheral receptor) was
first cloned and characterized for expression by Munro and coworkers (73). The CB;
receptor is predominantly expressed in cells of the immune system (74, 75) and has been
thought to mediate many of the immune suppressive effects of cannabinoids. In

particular, the CB; receptor has been shown to mediate the cannabinoid-induced

l8

inhibition of antigen processing and presentation in macrophage (76, 77), and antitumor
activity in a murine model of lung cancer (78). These studies illustrate different aspects

of host immunity that are cannabinoid receptor (CB; and CB;)-dependent

2) Alternative receptors

In addition to CB;/CB; receptor-dependent interactions, cannabinoids have also
been reported to induce CB;/CB; receptor-independent effects. Independence of the
CB;/CB; receptors has been evidenced in many different cells of the body (79—81). Of
particular interest to this dissertation are the CB ;/CB; receptor-independent effects
exhibited by immune cells. There is emerging evidence for cannabinoid-mediated effects
on cytokine production pertinent to T-dependent humoral and cell mediated immune
responses that occur independently of CB;/CB; receptors. Specifically, the suppression of
IL-2 production by cannabinoid treated mouse splenocytes stimulated with phorbol
myristate acetate/ionomycin (PMA/Io) could not be attenuated by the cannabinoid
receptor antagonists, SR141716A and SR144528 (82). Moreover, it has been
demonstrated that the endocannabinoid, 2-arachidonyl glycerol, suppressed PMA/Io
elicited IFN-y expression Similarly in splenocytes attained from cannabinoid receptor-
null mice (CB;'/'/CB;'/') as compared to splenocytes from wild type mice (83). Additional
evidence for cannabinoid receptor-independent effects of cannabinoids are illustrated by
the plant-derived cannabinoid, cannabidiol. Cannabidiol possesses little to no affinity for
the cannabinoid receptors CB; and CB;, yet it exhibits potent anti-inflammatory activity
including the reduced production of prostaglandin E2, nitric oxide and other oxygen-

derived free radicals to carrageenan-induced inflammation (84), inhibition of the release

19

of IL-1, TNF-a, and IFN-y by peripheral blood mononuclear cells (85), and suppressed
MIP-1a, MIP-1B, and IL-8 in the HTLV-l B cell line (86). To rigorously examine the
combined functional contribution of the CB;/CB; receptors to biological responses
elicited by cannabinoids, mice were developed with specific mutations in the CB; and/or
CB; receptor (77, 87).

Zimmer et al., (1999) (87) generated CB;'/' mice by replacing the coding region in
the mouse CB; receptor gene, between amino acids 32 and 448, with a PGK-neo cassette,
thereby creating embryonic stem cells with a mutant CB; gene. These cells were used to
create chimeric mice, which were bred with C57BL/6J mice. The resulting heterozygous
CB;+/- mice were then inbred to produce CB;'/' and CBC”+ mice. In a Similar fashion, the
CB;'/’ mouse was generated (77) and subsequently CB;'/' / CB;'/' mice were generated by

breeding both receptor-null mutants.

D) Effects of cannabinoids on the immune system

Cannabinoid receptors CB; and CB; and their transcripts have been identified on
cells involved in immune responses, including: B cells, T cells, monocytes, PMNS, and
NK cells (75, 88). Hence, through interactions with CB; and CB; receptors on immune
cells, cannabinoids have the potential to alter both innate and acquired immunity toward

invasive pathogens.
1) Effects on macrophage function

Macrophages are components of innate, cell-mediated, and humoral immunity.

Macrophages can circulate as blood monocytes or take residence in the liver as Kuppfer

20

cells or the lung as alveolar macrophages. Their phagocytic properties allow for effective
removal of circulating debris or xenobiotics. In addition, macrophages release an arsenal
of chemical mediators including nitric oxide that are toxic to bacteria and other human
pathogens.

Although the mechanism has not been fully defined, cannabinoids alter
macrophage function in vitro and in vivo. Macrophages treated in vitro to graded doses of
A9-THC have been Shown to suppress the ability of these cells to spread and to
phagocytose particles (89). In addition, exposure of mouse macrophage and human
peripheral blood monocyte preparations to Ag-THC led to decreases in TNF-a production
in response to lipopolysaccharide and IFN-y (90). Likewise, A9-THC has been shown to
inhibit nitric oxide production by mouse macrophages in a concentration and time-
dependent manner (91), and the release, but not synthesis, of IL-1 from peritoneal
macrophage cultures stimulated with endotoxin (92).

Macrophages are also antigen-presenting cells of the humoral arm of immunity. In
brief, antigen-presenting cells internalize antigen, proteolytically cleave the antigen and
present the antigen in the form of a complex with MHC class II molecules at the cell
surface. Recognition of MHC class II molecules by CD4+ T cells in the presence of co-
stimulatory Signals leads to activation and clonal expansion of T cells. Cannabinoids, in
particular Ag-THC, have been Shown to interfere with the ability of macrophages to

process antigen (93).

2) Effects on neutrophil function

Once the macrophage has encountered an invading pathogen such as bacteria it

21

can release cytokines to enhance vascular permeability and chemokines to direct the
migration of leukocytes. Neutrophils, or white blood cells, circulating in the blood are
one of the first cells recruited during an infection. Neutrophils roll along endothelium that
expresses cellular adhesion molecules, and then by diapedesis make their way into the
tissue where they can either phagocytose xenobiotic particulates or secrete proteases or
reactive oxygen species directed at killing the invading pathogen.

Smith and coworkers (94) examined cannabinoid receptor ligands for their effects
on the development of peritoneal inflammation elicited in mice with either thioglycollate
broth or staphylococcus enterotoxin A. The synthetic cannabinoid receptor agonists, HU-
210 and WIN 55212-2, blocked the migration of neutrophils into the peritoneal cavity in
response to these inflammatory stimuli. The authors attributed the effects to a delay in the
production of the neutrophil chemoattractant, kuppfer cell and macrophage inflammatory

protein-2 (MIP-2).

3) Effect on natural killer (N10 cell function

NK cells are large granular lymphocytes important in host resistance to tumor
cells and virally infected cells. NK cells target host cells with decreased expression of
MHC class 1 surface molecules in the presence of an activating co-stimulatory ligand.
When activated, NK cells release perforin and granzyme, which puncture holes into
target cells and initiate apoptotic cell death. NK cell activity can be augmented by
cytokines such as IL-2.

Lymphokine activated killer cells (LAK), generated by preincubation of NK cells

with IL-2, exposed to A9-THC at various concentrations exhibit decreased cytolytic

22

activity toward erythroleukemia (EL-4) target tumor cells, which are highly susceptible to
LAK cell killing. Conversely, the killing of YAC-l cells, a non-IL-2-dependent NK cell

target, was less affected by treatment with A9-THC (95).

4) Effects on T ”1 cells

A T cell-dependent cell-mediated immune response involves the destruction of
intracellular pathogens by macrophages activated by THl cells, or through the killing of
infected cells by cytotoxic CD8+ T cells. Regardless, both mechanisms target cells
infected with intracellular pathogens.

Clonal expansion and differentiation is a critical step for T cell responses to an
activating Signal. The expansion of T cells in response to mitogens or other ligands
requires the synthesis of IL-2 and the expression of IL-2 receptors on T cells. A9-THC
and 11-hydroxy-A9-THC inhibit the lymphoproliferative response to T cell mitogens (96).
Accordingly, Ag-THC has been Shown to inhibit IL-2-driven proliferation of T cells in a
dose-dependent manner (97). In addition, A9-THC has been Shown to decrease the surface
expression of high and intermediate-affinity IL-2 receptors, while increasing the
expression of low-affinity IL-2 receptors on the surface of T cells (98).

Differentiation of THO into THl or TH2 cell types is dependent on IL-12 or IL-4,
respectively. In a mouse model for cell-mediated immunity utilizing the bacterial
pathogen Legionella pneumophila, Klein and coworkers (2000)(99) reported that A9-THC
suppressed TH] immunity by attenuating the induction of THI-promoting cytokines such
as IFN-y, IL-12, and the receptor IL-12RB2. Therefore, the host could not develop an

appropriate TH] cell-mediated response to deal with the infecting pathogen.

23

5) Effects on CD8+ T cells

Cytotoxic T lymphocytes (CTL) are CD8Jr T cellS that provide protective
immunity against viral infection and intracellular pathogens. CTL’S mediate protective
immunity by inducing the apoptotic cell death of the infected target cell. Apoptosis is
induced either by Fas-mediated cell death or perforin and granzyme-mediated target-cell
killing.

F ischer-Stenger (100) examined the effect of Ag-THC on T cell functional
competence against Herpes Simplex Virus type 1 (HSVl) infection. Cytotoxicity assays
demonstrated that CTL from mice exposed to A9-THC were deficient in anti-HSVI
cytolytic activity. A9-THC in vivo exposure affected CTL cytoplasmic polarization
toward the virus-infected target cell resulting in a lower frequency of CTL granule
reorientations toward the effector cell-target cell interface following cell conjugation.

Hence, there was decreased cell-mediated immunity through CTL activation.

6) Effects on immune cell-derived chemokines and cytokines
Chemokines and cytokines produced by leukocytes in response to inflammatory
stimuli provide the necessary Signaling to support leukocyte function in the defense
against an invading pathogen. Cannabinoids have been shown to suppress the production
of chemokines including IL-8, MIP-la, MIP-lB, RANTES and MCP-l (86, 94). In
addition, cannabinoids have been shown to modulate the expression of a wide range of
cytokines including IFN-y, transforming growth factor-beta (TGF-B), TNF-a, IL-2, IL-4,

IL-6, IL-10, IL-12, IL-13, and IL-15 in human and murine cell models (101). The

24

implication of these findings in the context of pathogen challenge is that cannabinoid
treatment would suppress the recruitment of leukocytes and fiuther suppress the ability of
T cells and macrOphageS to communicate effectively when mounting cell-mediated or
humoral immune responses.

Regulation of the production of these cytokines by cannabinoids has been linked
with the suppression of DNA-binding activity of transcription factors. In particular, AP-
], NF-KB, STATS, and the nuclear factor of activated T cells (N F-AT). A9-THC has been
shown to inhibit PMA/ionomycin—induced AP-l DNA-binding activity (102), the
activation and binding of NF -l<B/Rel proteins to their DNA binding Site (103), and the
tyrosine phosphorylation of STAT-1 alpha protein (104). More recently, the
endocannabinoid 2-arachidonoyl-glycerol has also been shown to disrupt nuclear

translocation of NF-AT protein (83).

E) Models of host-resistance

Animal models of host resistance are important for elucidating whether exposure
to specific chemicals recognized for their effects on host-immunity, like A9-THC, render
the host more susceptible to pathogenic challenge. A9-THC has been demonstrated to
have an effect on host-resistance to a variety of pathogens including Herpes Simplex
virus (100) and Legionella pneumophila (105). In these models, it has been Shown that
A9-THC modulates T cell functionality, thereby suppressing normal pathogen clearance

mechanisms.

1) Model of A9-THC and Listeria Monocytogenes

25

Listeria Monocytogenes is a gram-positive bacterium that causes the foodbome
illness listeriosis. Listersiosis is associated with sepiticemia and meningitis in humans.
Morahan et a1. (1979) (106) examined bacterial infection in BALB/c mice at a 50% lethal
dose alone and in combination with A9-THC treatment. A9-THC was dissolved in a
mixture of Emulphor and ethanol and administered intraperitonealy at a dose of 200
mg/kg on the first 2 days of infection. The bacterium was delivered by intravenous
inoculation. Mortality of the mice was used as an endpoint and the lethal dose to 50% of
the population (LD50) was calculated. A9-THC treatment decreased host-resistance to
infection as calculated by the arithmetic difference between LD50 values of the untreated
and treated groups. The authors concluded that A9-THC suppressed cell-mediated
immunity, and suggested that the suppression of cell-mediated immunity was likely the

result of an effect on T cells by A9-THC.

2) Model of A9-THC and Herpes Simplex Virus

Herpes Simplex Virus type 2 (HSV-2) is an infection that leads to the formation
of blisters in the skin or mucous membranes. It is typically transmitted via the lips or
genitals. When the virus is asymptomatic it lies dormant in nerve cells. In an experiment
of similar design to that of the bacterium Listeria Monocytogenes, Morahan et a1 (1979)
(106) infected BALB/c mice with an LD50 dose of HSV-2 by intravenous inoculation.
Again, A9-THC was dissolved in a mixture of Emulphor and ethanol and administered
intraperitonealy at a dose of 200 mg/kg on the first 2 days of infection. Mortality of the
mice was used as an endpoint. A9-THC decreased host-resistance to infection as

calculated by the arithmetic difference between LD50 values of the untreated and treated

26

groups. The collective conclusion offered by the authors was that Ag-THC treatment
decreased cell-mediated immunity.

Mishkin and coworkers (1985) (107) examined the effects of A9-THC treatment
on vaginal infections in B6C3F1 mice. A9-THC was dissolved in Emulphor and ethanol
and administered by intraperitoneal injection at doses ranging from 15 to 100 mg/kg for 4
consecutive days starting on the day before intravaginal infection with HSV-2. The
authors concluded that A9-THC decreased resistance to HSV-2 in a dose-related manner
with an increased trend toward cumulative mortalities and acceleration of the time-to-
onset of death.

In 1992, Fischer-Stenger and coworkers (100) examined the effect of Ag-THC on
cytolytic T cell function against HSV-l infection. Specifically, C3H/HeJ mice were
treated with A9-THC at doses 15 or 100 mg/kg in a mixture of Emulphor and ethanol
intraperitonealy on days 1-4, 8-11, and 15-18. Mice received an intraperitoneal injection
of 1x 107 plaque forming units (pfu) of HSVl on day 21. Spleens were removed 7 days
later and T cells were examined by flow cytometry and Nomarski Microscopy. The
authors concluded that Ag-THC treatment decreased cytolytic activity by altering the
polarization of the T cells. The inability to polarize rendered the T cells incapable of
mounting an efficient release of granules containing the pore-forming protein perforin to

the infected cells.

3) Model of A9-THC and Legionella pneumophila
Legionella pneumophila is a gram-negative bacterium that causes Legionnaires'

disease. The primary Site of infection is the respiratory tract where it causes pneumonia.

27

Legionella pneumophila is an intracellular parasite that infects macrophages after it has
been phagocytosed. Therefore, the macrophage is not capable of destroying the pathogen.

Klein et a1. (1993) (105) treated BALB/c mice with A9-THC dissolved in
dimethyl sulphoxide and heat inactivated mouse serum. Mice were injected intravenously
with 8 mg/kg A9-THC 24 hours prior to infection and again 24 hours post infection. The
bacterium was delivered at a sublethal concentration by intravenous inoculation.
Mortality of the mice occurred within 30 minutes of the second injection of A9-THC.
Treatment of mice with an antibody to IL-6 or TNF-a had a protective effect against
mortality. Hence, the authors concluded that the mice died from shock.

In a subsequent study by Klein and coworkers (2000) (99), the effects of A9-THC
in modulating cytokines involved in the development of THI cells were examined. The
study employed a Single injection of A9-THC intravenously at a dose of 8 mg/kg, 18
hours prior to infection with Legionella pneumophila. The study revealed that treatment
of mice with A9-THC increased the production of the cytokine IL—4, but suppressed IL-12
and IFN-y serum levels. By implementing cannabinoid receptor (CB; and CB;)
antagonists into the study, it was demonstrated that both the CB; and CB; receptors
might be involved.

In a more recent study, Lu et al., (2006) (108) demonstrated that IL-12
suppression by A9-THC was, in part, independent of the CB; and CB; receptor. A9-THC
suppression of IL-12 production in dendritic cells obtained from receptor knockout mice
was only partially attenuated in the absence of receptors. Moreover, Lu et al (2006b)
(109) demonstrated that A9-THC inhibits THl activation by targeting dendritic cell

maturation and. expression of co-stimulatory and polarizing molecules.

28

IV. Objectives

The objective for the studies outlined in this dissertation project was to examine
the hypothesis that the airways of mice treated with A9-THC are more susceptible to the
adverse sequelae of influenza virus in a CB;/CB;-receptor-independent manner. First, the
magnitude and severity of inflammation associated with the dose of influenza virus alone
was established. Second, two different doses of A9-THC were administered to mice for
five days by oral gavage and blood serum was collected to establish the steady state
blood serum concentration of A9-THC and its metabolites. Third, the time-dependent
effects of influenza on immune and epithelial cell responses to pulmonary infection with
influenza were assessed to provide the kinetic basis for monitoring critical time points for
A9-THC-related effects. Fourth, the dose-dependent effect of A9-THC on immune and
epithelial cell responses was evaluated. Fifth, the immune and epithelial cell effects of
A9-THC were examined in the context of mice deficient in the cannabinoid receptors CB;
and CB;. The results of these studies demonstrated that there was a classical pattern of
inflammation induced by influenza challenge with concurrent time-dependent changes in
epithelial cell death, regeneration, and mucous cell metaplasia. A9-THC increased viral
H1 mRNA levels in the lung with a concomitant decrease in macrophages and
lymphocytes that are essential for the effective cell-mediated clearance of virus infected
epithelial cells. Treatment of CB;"'/CB;‘/' mice with A9-THC led to alterations in viral H1
mRNA levels, immune cell function, and concomitant morphologic changes of the
airway epithelium that were both Similar for and unique to CB;'/'/CB;'/' mice as

compared to wild type mice.

29

MATERIALS AND METHODS
I. Cannabinoid compounds
A9-THC was provided by the National Institute on Drug Abuse (Bethesda, MD)

as a resin. The resin was solubilized in corn oil for administration to mice by oral gavage.

11. Animals

C57BL/6 mice (8-10 weeks old), free of pathogens and respiratory disease, were
purchased from Charles River (Portage, MI). On arrival, mice were randomly assigned to
their experimental groups, transferred to plastic cages containing sawdust bedding, given
food (Purina Certified Laboratory Chow) and water ad libitum, and quarantined for 1
week until their body weight was 17—20 g. Mice were used in accordance with guidelines
set forth by the Institutional Animal Care and Use Committee at Michigan State
University. Animal holding rooms were maintained at 21—24°C and 40—60% relative

humidity with a 12-h light/dark cycle.

III. Experimental designs

Five different study designs were employed. The first study was conducted to
identify a concentration of PR8 to be administered intranasally to mice that would
produce a mild inflammatory response in the lungs by 13 days post infection (dpi)
without mortality of the mice (Appendix 1). The second study examined the blood levels
of A9-THC and its metabolites following five consecutive days of dosing with A9-THC by
oral gavage (Appendix 2). The third study addressed time-dependent changes in the

pulmonary response to PR 8 challenge (Appendix 3). The fourth study evaluated the

30

effects of varying concentrations of A9-THC on the previously characterized time-
dependent changes in immune and epithelial cell responses to PR8 (Appendix 4). Finally,
the fifth study examined the role of the cannabinoid receptors (CB; and CB;) in
mediating the effects of A9-THC on immune and epithelial cell responses following PR8

challenge (Appendix 5).

IV. Influenza A/PR/8/34 instillation

Influenza A/PR/8/34 (PR8) was generously supplied by the laboratory of Dr. Alan
Harmsen (Montana State University, MT). Mice were anesthetized with 4% isoflurane in
oxygen, and 50 ul of PR8 in pyrogen-free SAL (SAL) was instilled as 25 ul per nare at a

total dose of 50 plaque forming units (pfu).

V. Steady state blood serum levels of A9-THC and its metabolites.

The Ag-THC utilized in these studies came from a resin that was first weighed
and then solubilized in corn oil at different dilutions to give final concentrations of either
25 mg/kg, 50 mg/kg, or 75 mg/kg. A9-THC was administered by oral gavage to C57BL/6
mice for five consecutive days. On the fifth day, blood was taken by cardiac puncture 4 h
after the final dose of A9-THC. Blood was stored in red vaccutainers on ice and shipped
to the laboratory of Dr. Jim Klaunig for the determination of blood serum levels of A9-

THC and the primary metabolites, 9-COOH-A9-THC, and ll-OH-A9-THC.

VI. Necropsy, lavage collection, and tissue preparation

On the day sacrifice, mice were anesthetized by an ip injection of 0.1 ml of 12%

31

pentobarbital solution, a midline laparotomy was performed, and Mice exsanguinated by
cutting the abdominal aorta. Immediately after death, the trachea was exposed and
cannulated, the heart and lung were excised en bloc. One milliliter of sterile SAL was
instilled through the tracheal cannula and withdrawn to recover bronchoalveolar lavage
fluid (BALF). A second SAL lavage was performed and combined with the first.

After lavage, the lung was processed for histological analysis. The left and right
lung lobes were inflated under constant pressure (30 cm H20) with 10% neutral buffered
formalin (Sigma Chemical Co., St. Louis, M0) for 1 h. The tracheal airway was ligated
and the inflated lobes were stored in a large volume of the same fixative for at least 24 h
until further processing.

The intrapulmonary airways of the fixed left or right lung lobes from each rodent
were microdissected according to a modified version of the technique of Plopper et
al.(l983) (110) and fully described in a previous publication (111). Beginning at the lobar
bronchus, airways are split down the long axis of the largest daughter branches (i.e., main
axial airway; large diameter conducting airway) through the twelfth airway generation.
Two transverse tissue blocks were excised at the level of the fifth (proximal) and eleventh
(distal) airway generation (Figure 3). Transverse tissue blocks were also excised from the
middle of the four right lung lobes perpendicular to the largest airway branch entering
each lobe (Figure 3). Tissue blocks from the left and right lung lobes were embedded in
paraffin, sectioned at a thickness of 5 pm, and then stained with hematoxylin and eosin
(H&E) for light microscopic examination. Other paraffin sections were stained with
either alcian blue/ periodic acid Schiff (AB/PAS) to detect neutral and acidc

mucosubstances or alcian blue (pH 2.5)/Hematoxylin (AB/H) to identify acidic

32

Right lung R1
lobes Left lung lobe

 

y‘ 65 II

 

If
>

 

v

 

 

R3 \ 611%

‘i\

Figure 3. Diagram of the microdissection of mouse right lung lobes (R1, R2, R3, R4)
and left lung lobes at generations 5 (G5) and 11 (G11).

33

intraepithelial mucosubstances.

VII. Immunocytochemistry.

Hydrated paraffin sections (5—6 um thick) from formalin-fixed lung tissues were
treated with 0.05% proteinase K for 2 min and washed with l N HCI for 1 h. Sections
were then treated with 3% H;0; (in methanol) to block endogenous peroxide and were
incubated with a monoclonal antibody (PC10) cocktail to PCNA (Biogenex, San Ramon,
CA) consisting of the primary antibody to PCNA 1:50, a secondary antibody to
Immunoglobulin 1:500 and mouse serum 1:50 for 1 h. Immunoreactive PCNA was
visualized with the Vectastain Elite ABC kit (Vectastain Laboratories Inc., Burlingame,
CA) using 3‘,3‘-diaminobenzidine (DAB) tetrahydrochloride (Sigma Chemical Co., St.

Louis, MO) as a chromagen.

VIII. Total and PCNA positive epithelial numeric cell density and labeling index.
The total number of epithelia lining the luminal surface of the main axial airway
at generation 5 was enumerated per length of basal lamina. Likewise, cells with nuclei
staining positive for PCNA were also enumerated per length of basal lamina. A labeling
index for PCNA was determined by dividing the number of PCNA positive cells per unit
length of basal lamina by the total number of epithelial cells per unit length of basal

lamina.

IX. CAS-3 and alcian blue numeric cell densities. Slides of lung sections either stained
immunohistochemically for CAS-3 or stained for alcian blue (acidic mucosubstances)

were examined. Numeric cell densities were determined for epithelial cells

34

immunohistochemically reactive to CAS-3 via light microscopy by counting the number
of nuclear profiles of these immunoreactive epithelial cells lining the bronchiolar
epithelium at generation 5 and dividing by the length of the underlying basal lamina.
Numeric cell densities for CAS-3 were expressed as the number of immunoreactive cells
per mm basal lamina. In a Similar manner, numeric cell densities were determined for
epithelial cells staining with alcian blue (acidic mucosubstances). The numeric cell
density of epithelial cells staining for alcian blue (acidic mucosubstances) was expressed

as the number of alcian blue reactive epithelial cells per mm basal lamina.

X. Morphometry of stored intraepithelial mucosubstances.

The volume density (Vs) of AB/PAS-Stained mucosubstances in the respiratory
epithelium lining the main axial airway at 3, 7, 10, 15, and 21 dpi was quantified using
computerized image analysis and standard morphometric techniques. The area of
AB/PAS stained mucosubstance was calculated from the automatically circumscribed
perimeter of stained material using a Power Macintosh 7100/66 computer and the public
domain NIH Image program (written by Wayne Rasband, US. National Institutes of
Health and available on the Internet at http://rsb.info.nih.gov/nih-image/l The length of
the basal lamina underlying the surface epithelium was calculated from the contour
length of the digitized image of the basal lamina. The volume of stored mucosubstances
per unit of surface area of epithelial basal lamina was estimated using the method
described in detail by Harkema et al., (1987) (112). The Vs of intraepithelial
2

mucosubstances was expressed as nanoliters of intraepithelial mucosubstances per mm

of basal lamina.

35

XI. Bronchoalveolar lavage cellularity.

The total number of leukocytes in BALF was counted with a hemacytometer. In
brief, 10 ul of BALF was added to a hemacytometer and the number of leukocytes in the
four etched comer quadrants were counted and multiplied by 2500 to yield the number of
leukocytes per ml of sample. The percent of total leukocytes consisting of eosinophils,
lymphocytes, macrophages, and neutrophils were determined from counts of 200 cells in
a cytospin sample stained with Diff-Quick (Dade Behring, Newark, DE). The percentage
of eosinophils, lymphocytes, macrophages, and neutrophils were multiplied by the total
number of leukocytes determined from the hemacytometer to yield the respective number

of each cell type per ml of sample.

XH. Histopathology scores for inflammation.

A histopathologic score was established based upon the numbers and distribution
of inflammatory cells within the tissues, as well as non-inflammatory changes such as
evidence of bronchiolar epithelial injury and repair. The scores assigned were: 0 = no
inflammation, 1 = mild, inflammatory cell infiltrate of the perivascular/peribronchiolar
compartment, 2 = moderate, inflammatory cell infiltrate of the
perivascular/peribronchiolar space with modest extension into the alveolar parenchyma,
and 3 = severe, inflammatory cell infiltrate of the perivascular/peribronchiolar space with
a greater magnitude of inflammatory foci found in the alveolar parenchyma. A certified
pathologist scored each lung section independently and a mean score with the standard

error of the mean was calculated for each treatment group.

36

XIII. Total protein.

Total protein was quantified in BALF using the bicinchoninic acid (BCA) method
as provided by the manufacturer (Pierce, Rockford, IL). In brief, BALF samples were
centrifuged at 600 rpm for 5 min to pellet cellular debris. Supernatants were removed and
stored at -20°C until testing was performed. 25 31 of BALF supernatant or bovine serum
albumin protein standard (25-2000 [jg/ml) was incubated in a 96-well microplate with
200 131 of a 50:1 mixture of reagents A (sodium carbonate, sodium bicarbonate,
bicinchoninic acid and sodium taltrate in 0.1M sodium hydroxide) and B (4% cupric
sulfate) for 30 minutes at 37°C. Samples were read on a Biotek microplate reader at 562

nm.

XIV. Neutrophil elastase.

Airway elastase recovered in BALF was determined by an ELISA for elastase
using a rabbit monoclonal antibody to the human elastase (Calbiochem, La Jolla, CA). 50
ul aliquots of BALF were applied to a 96-well microtiter plate (Microfluor 2 Black,
Dynex Technologies, Chantilly, VA) and dried overnight at 40°C. Plates were blocked
with a solution of 1.5% goat serum in automation buffer solution (ABS, pH 7.5; Biomeda
Corp., Foster City, CA) for 30 min at 37°C. Plates were then incubated with anti-elastase
antibody (1:400 in ABS containing 1.5% goat serum) for l h at 37°C and then washed
three times with ABS. Bound primary antibody was detected with a biotinylated goat
anti-rabbit secondary antibody and quantitated using horseradiSh-peroxidase-conjugated
avidin/biotin complex (ABC Reagent; Vector Laboratories, Burlingame, CA) and a

fluorescent substrate (QuantaBlue; Pierce Chemical, Rockford, IL) using a fluorescence

37

microplate reader (SpectraMax Gemini; Molecular Devices; 318 nm excitation/410 nm
emission). Readings were taken at 3 min intervals for 24 min. Duplicate samples were

averaged and the group data is represented as mean Vmax units/S.

XV. Inflammatory and T;;1/T;;2 cytokines. BALF samples were centrifuged at 600
rpm for 5 min to pellet cellular debris. Supernatants were removed and stored at -20°C
until testing was performed. The cytokines IL-6, IL-10, MCP-l, IFN-y, TNF-a, and IL-
l2p70 were detected Simultaneously by using the cytometric bead array (CBA) mouse
inflammation kit (BD Pharmingen, San Diego, CA) and the THl/I‘H2 cytokines IL-2, IL-
4, IL-5, IFN-y, and TNF-a were detected simultaneously by using the mouse THl/THZ
kit. In brief, 50 ul of BALF or serum from each sample was incubated individually with a
mixture of capture beads and 50 ul of PE detection reagent consisting of PE-conjugated
anti-mouse IL-6, IL-10, MCP-l, IFN-y, TNF-a, and IL-12p70 or 50 ul of PE detection
reagent consisting of PE-conjugated anti-mouse IL-2, IL-4, IL-5, IFN-y, and TNF-a. The
samples were incubated at room temperature for 2 h in the dark. After incubation,
samples were washed once and resuspended in 300 pl of wash buffer before acquisition
on a BD FACSCalibur flow cytometer. Data were analyzed using CBA software (BD
Pharmingen, San Diego, CA). Standard curves were generated for each cytokine using
the mixed cytokine standard provided with the respective kits. The concentration of each
cytokine was determined by interpolation from the corresponding standard curve. The
range of detection was 20 — 5000 pg/ml for each cytokine measured. Serum was analyzed
for inflammatory cytokines only.

In a similar manner, the cytokines IL-9 and IL-13 were analyzed using BD flex-

38

sets. Samples and standards were analyzed according to manufacturer-based instructions.

XVI. T-Iymphocyte flow cytometry. After enumerating the retrieved cells in BALF, the
cells were pelleted by centrifugation and reconstituted in 150 pl flow cytometer (FCM)
buffer (PBS supplemented with 2% (w/v) bovine serum albumin and 0.09% (w/v) sodium
azide) with purified anti-mouse CD16/CD32 (Fey III/II Receptor) antibody (BD
Biosciences, San Jose, CA) to block for 30 min. The samples were then Split into two
groups of equal volume, washed and reconstituted in FCM buffer. One group received
antibodies for CD3 (APC anti-mouse CD38), CD4 [PE anti-mouse CD4(L3T4)] and CD8
[FITC anti-mouse CD8a(Ly-2)], while the other group received the cognate isotype
control antibodies. Samples were allowed to incubate l h at 4°C, washed twice and then
fixed for 10 min. with Cytofix. Samples were then washed again and reconstituted to a
volume of 300 pl for analysis on the BD FACSCalibur flow cytometer. The total number

of events taken per each sample was 10,000.

XVII. RNA isolation.

Total RNA was isolated from the lung lobes by using the TRI-reagent method
(Sigma Chemical, St Louis, MO). The evaluation of the relative expression levels of H1,
Caspase-3 and MUCSAC messenger ribonucleic acid (mRNA) were determined using the
TaqMan real-time multiplex reverse transcriptase-polymerase chain reaction (RT-PCR)
with custom designed TaqMan primers and probe to the target gene and the

manufacturer’s pre-developed primers and probe to 183 (Applied Biosystems, Foster

39

City, CA). The primers and probe to both the target gene and endogenous reference gene
were Specifically designed to exclude detection of genomic deoxynucleic acid (DNA).
Aliquots of isolated tissue RNA (1 ug total RNA) were converted to copy DNA (cDNA)
using random primers. The resultant cDNA (2 pl) was added to a reaction mixture that
consisted of the target gene primers and probe, endogenous reference primers and probe
(188 ribosomal RNA), and Taqman universal master mix to a final volume of 30 ul.
Following PCR, amplification plots (change in dye fluorescence versus cycle number)
were examined and a dye fluorescence threshold within the exponential phase of the
reaction was set separately for the target gene and the endogenous reference (188). The
cycle number at which each amplified product crosses the set threshold represents the CT
value. The amount of target gene normalized to its endogenous reference was calculated
by subtracting the endogenous reference C; from the target gene CT (ACT). Relative
mRNA expression was calculated by subtracting the mean AC; of the control samples
from the AC; of the treated samples (AACT). The amount of target mRNA, normalized to
the endogenous reference and relative to the calibrator (i.e., RNA from control) is

calculated by using the formula Z‘MCT.

XVIII. Statistical Analysis.

Data are expressed as mean is standard error of the mean (SEM). The differences
between treatment groups were determined by either a Student's t-test, one-way or two-
way analysis of variance (ANOVA) with multiple comparisons made by the Student-
Newman-Keuls post hoc test using SigmaStat software from Jandel Scientific (San

Rafael, CA). The criterion for significance was taken to be p <0.05.

40

EXPERIMENTAL RESULTS

1. Model development: Establishing the dose.

A) Concentration-dependent inflammatory responses to PR8 in pulmonary

airways

The inflammatory response to PR8 was confined to the hilar region of the
transverse left lung lobe sections (Figure 4). The inflammatory response was most
notable at generation 5 with occasional evidence of inflammation observed at generation
11. The magnitude and severity of the inflammatory response was concentration-
dependent, increasing with increasing concentrations of PR8. The inflammatory response
consisted primarily of mononuclear cells that included lymphocytes,

monocytes/macrophages, and neutrophils.

B) Steady state blood serum levels of A9-THC and its metabolites.

The steady state blood serum levels for A9-THC and its primary metabolites, 9-
COOH-Ag-THC, and ll-OH-A9-THC were determined on the fifth day of treatment, 4 h
after the final dose of Ag-THC (Table 1). At a dose of 5 mg/kg A9-THC, blood serum
levels reached a mean concentration of 84.6 ng/ml Ag-THC. In addition, there was
evidence of modest levels of the 9-COOH-A9-THC metabolite present in blood serum
with no detectable levels of the ll-OH-A9-THC metabolite. At a dose of 75 mg/kg A9-
THC, the mean blood serum levels for the parent compound were similar to those
observed with the 5 mg/kg A9-THC dose. However, levels of the 9-COOH-A9-THC
metabolite in blood serum were augmented when compared to the blood serum levels for

mice receiving 5 mg/kg A9-THC with concentrations reaching 446 ng/ml 9-COOH-A9-

41

Figure 4. Increasing concentrations of influenza virus result in increasing
inflammatory responses in the hilar region of the left lung lobe at generation 5 of the
main axial airway. Light photomicrographs of the respiratory epithelium lining the
luminal surface of the main axial airway (MAA) (generation 5) from mice intranasally
instilled with PR8 at concentrations of 50 pfu (A), 300 pfu (B), and 500 pfu (C) at 13
days post infection. There was marked inflammatory cell infiltration of the
perivascular/peribronchiolar submucosa (sm) extending into the alveolar parenchyma (p)
in all of the sections representing each concentration of virus. There was a concentration-
dependent increase in the magnitude and severity of the inflammatory response observed.

Artery = (a). bar = 100 microns. Images in this dissertation are presented in color.

42

 

Figure 4. Increasing concentrations of influenza virus result in increasing
inflammatory responses in the hilar region of the left lung lobe at generation 5 of the

main axial airway.

43

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample # A9-THC 9-coon-A9- ll-OH-Ag-THC
Vehicle (ng/ml) THC (ng/ml) (ng/ml)
1 3.6 ND ND
2 2.5 ND ND
3 3.1 ND ND
4 42 ND ND
mean 3.4
Sample # A9-THC 9-COOH-A9- ll-oH-A9-THC
5mg/kg THC (ng/ml) THC (ng/ml) (rig/ml)
1 55.2 6.8 ND
2 48.4 24.6 ND
3 133.5 30.0 ND
4 101.4 41.0 ND
mean 84.6 25.6
Sample # A9-Tnc 9-cooH-A9- 11-0H-A9-Tllc
75mg/kg THC (ng/ml) THC (ng/ml) (ng/ml)
1 29.9 136.8 2.7
2 64.5 873.3 193
3 117.9 317.0 10.1
4 52.4 459.0 9.9
mean 66.2 446.5 10.5

 

 

Table 1. Analysis of A9-THC, 9-C00H-A9-THC, and ll-OH-A9-THC
concentrations in mouse blood serum. C57BL/6 mice were treated with vehicle (corn
oil), 5 mg/kg or 75 mg/kg A9-THC for 5 consecutive days. Four hours after the last
treatment, mice were anesthetized and whole blood was collected by cardiac puncture.

A9-THC and its metabolites were chemically extracted from whole blood and analyzed by

GC/MS. ND = not detected.

44

THC. Lastly, the ll-OH-Ag-THC metabolite was modestly elevated with the 75 mg/kg

A9-THC treatment group with concentrations averaging 10 ng/ml.

H. Time-dependent airway epithelial and inflammatory cell responses induced by
influenza virus A/PR/8/34 in C57BL/6 mice.

A) PR8 induces time-dependent alterations in epithelial morphology

No microscopic alterations were present in the examined proximal (at GS of the
main axial airway) and distal (at G11 of the main axial airway) tissue sections from the
left lung lobe of mice that were intranasally instilled with vehicle alone (controls) (Figure
5A). The principal pulmonary alteration in mice intranasally instilled with influenza virus
and sacrificed 3 dpi (Figure 5 A-D) was an acute necrotizing bronchiolitis characterized
by multiple focal areas of necrosis and luminal shedding (exfoliation) of the surface
epithelial cells lining the main axial airway (Figure 5C) and smaller diameter pre-
terminal and occasional terminal bronchioles. Similar but much less severe lesions were
observed in the distal tissue section of some of these exposed mice. Airway epithelial
lesions were accompanied by a mild intramural inflammatory cell infiltrate composed
principally of mononuclear cells (lymphocytes and monocytes) and lesser numbers of
neutrophils. A Similar inflammatory cell infiltrate was present in adjacent peribronchiolar
and perivascular regions.

Virus instilled mice that were sacrificed 7 dpi had a marked bronchiolitis and
alveolitis again restricted mainly to the hilar region of the lung lobe (proximal tissue
section). Necrosis and exfoliation of the bronchiolar ciliated epithelium observed at 3 dpi

was replaced by a hyperplastic/hypertrophic, nonciliated, cuboidal and basophilic

45

Figure 5. Bronchiolar epithelial morphometry following influenza infection. Light
photomicrograph of the respiratory epithelium (e) lining the luminal surface of the main
axial airway (generation 5) from mice intranasally instilled with SAL (A, B, E, F, I, J)
and PR8 (C, D, G, H, K, L) at 3, 10, and 21 dpi, respectively. A = H&E; SAL instilled
mouse 3 dpi with no alterations to the respiratory epithelium lining the airway or to the
peribronchiolar tissue (a). B = AB/PAS; SAL instilled mouse 3 dpi with no microscopic
evidence of MCM. C = H&E; PR8 instilled mouse 3 dpi with degenerative respiratory
epithelium lining the airway and peribronchiolar inflammatory cell infiltrate in the
submucosa (sm). D = AB/PAS; PR8 instilled mouse 3 dpi with exfoliation of the
epithelium lining the luminal surface of the main axial airway and no microscopic
evidence of MCM. E = H&E; SAL instilled mouse 10 dpi with no alterations to the
respiratory epithelium lining the airway or to the peribronchiolar tissue. F = AB/PAS;
SAL instilled mouse 10 dpi with no microscopic evidence of MCM. G = H&E; PR8
instilled mouse 10 dpi with regenerative epithelium and mucous cell metaplasia (arrows)
with marked peribronchiolar inflammatory cell infiltrate (asterisk). H = AB/PAS; PR8
instilled mouse 10 dpi with marked accumulation of mucous-containing epithelial cells. I
= H&E; SAL instilled mouse 21 dpi with no alterations to the respiratory epithelium
lining the airway or to the peribronchiolar tissue. J = AB/PAS; SAL instilled mouse 21
dpi with no microscopic evidence of MCM. K = H&E; PR8 instilled mouse 21 dpi with
cuboidal epithelium and mucous cell metaplasia with decreased peribronchiolar
inflammation. L = AB/PAS; PR8 instilled mouse 21 dpi with marked accumulation of
mucous-containing epithelial cells lining the airway. Bar = 50 microns. Images in this

dissertation are presented in color.

46

 

 

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-.I \/."__§ m If a V’-
- o
- - 7 f-

...?" sm ,3"? v ' '

“-5’.l " T " «0"...

, I~ I a ‘—o. ’

 

 

e, F . . M -- v. -- : smrc ,7;
-kMid-bfifiu‘rfij .. _ I. \'l _
" - sm ,3 a v'
/ - x 7
sm V - d \y
— . — ., /—— - ~ ~.(_
I . I

Figure 5. Bronchiolar epithelial morphometry following influenza infection.

47

epithelium accompanied by a marked lymphocytic inflammatory cell infiltrate in the
affected bronchioles and surrounding alveolar parenchyma (interstitial pneumonia).
Lesser, but conspicuous, numbers of eosinophils were also intermixed with the
mononuclear inflammatory cells. There was also mild to moderate alveolar type II cell
hyperplasia and hypertrophy in these affected parenchyma] regions along with numerous
large highly vacuolated alveolar macrophages, smaller monocytes, and lymphocytes
within alveolar air Spaces. Various amounts of proteinacious material were also present in

some of the alveolar lumens in the affected regions of the pulmonary parenchyma.

Mice that were instilled with virus and sacrificed at 10 dpi (Figure 5 E-H) had a
chronic bronchiolitis and alveolitis, again restricted mainly to the proximal tissue section
(hilar aspect of the lung lobe). At 10 days post-infection, the affected bronchiolar
epithelium was composed of tall cuboidal to columnar ciliated and nonciliated cells
(Figure 5G). Many of the nonciliated epithelial cells were mucous cells with conspicuous
amounts of AB/PAS stained (Figure 5H), intracytoplasmic mucosubstances (i.e. Mucous
cell metaplasia). The associated inflammatory cell infiltrate in and around the bronchiolar
walls, adjacent blood vessels and alveolar parenchyma was similar in composition to that
observed in mice sacrificed at 7 dpi. However, the most conspicuous change in the
affected regions of the alveolar parenchyma at 10 dpi compared to that at 7 dpi was the
addition of coalescing regions of alveolar fibrosis accompanying the type 11 cell
hyperplasia and the mainly lymphocytic inflammatory cell infiltrate (chronic alveolitis).

Mice instilled with virus and sacrificed at 15 and 21 dpi (Figure 5 I-L) had Similar
but less severe airway and parenchyma] lesions as compared to those lungs of the mice

sacrificed at 10 dpi.

48

B) Temporal analysis of stored intraepithelial mucosubstances following PR8
challenge

The Vs of intracytoplasmic acidic and neutral mucosubstances (Figure 6) in the
airway epithelium was increased by approximately 4-fold in lung lobe sections obtained
from PR8 treated mice as compared to respective time-matched SAL controls starting as
early as 10 dpi. The marked elevation in mucosubstance Vs was maintained through 21

dpi.

C) Expression levels of whole lung MUCSAC mRNA at 7 days post infection

Recognizing that the time to onset of MCM was established at 10 dpi, RNA from
whole lung homogenates at 7 dpi was analyzed for MUCSAC mRNA levels (Figure 7).
MUCSAC levels were nearly 3-fold greater in lung homogenates from mice treated with

PR8 than those treated with SAL.

D) Time-dependent differences in total and PCNA immunopositive epithelial
numeric cell densities

By numeric cell density counts, the total airway epithelial cell counts (Figure 8A)
observed at 10 dpi were mildly elevated with respect to counts enumerated in SAL
controls. In addition to total cell counts, cells with nuclei staining positive for PCNA
(Figure 8B) were also enumerated. PCNA numeric cell density and labeling index
(Figure 8C) were Significantly elevated as compared to time-matched SAL controls by

four to six-fold in the PR8 treated group at 7 and 10 dpi, respectively.

49

 

 

 

1-5‘ 1:1 saline
x - Influenza
3g 1.2- * *
3 E
g g 0 8- *
3 .8
S E 0.4-
g E

0.0-

3 7 1O 15 21
Time (Days)

Figure 6. Time-to-onset of mucous cell metaplasia following influenza infection.
Effects of influenza instillation on the volume density of intraepithelial mucosubstances
in the epithelium lining the main axial pulmonary airways at generation 5 of the left lung
lobe. Mice were instilled with PR8 or SAL and sacrificed 3, 7, 10, 15, and 21 dpi and
tissues collected and processed as described in Materials and Methods. Data is expressed

as mean 3: SEM. * = significantly different from respective control instilled with SAL.

50

 

8
c: 4
«1
fi
1', 3
£
2 2
3 1
D
E

0

Saline Influenza
Treatment

Figure 7. MUCSAC gene induction by influenza at 7 days post infection. Effects of
influenza instillation on the levels of MUCSAC expression at 7 dpi in the lungs of mice
treated with either SAL or PR8. Data is expressed as mean 5: SEM. * = significantly
different from respective control instilled with SAL.

51

Figure 8. Time-dependent proliferation of epithelial cells lining the main axial
airway following influenza infection. Effects of influenza instillation on the numeric
cell density of total epithelial cells (A), PCNA positive cells (B), and the labeling index
for PCNA (C) in the epithelium lining the main axial pulmonary airways at generation 5
of the left lung lobe. Mice were instilled with PR8 or SAL and sacrificed 3, 7, 10, 15, and
21 dpi and tissues collected and processed as described in Materials and Methods. Data is
expressed as mean i SEM. * = significantly different from respective control instilled

with SAL.

52

13° [:1 Saline

I" a 150 - Influenza
3 .E
2 e 120
a a
g; 90
3' 60
35
e 30

o — _ —

3 7 1O 15 21
Time (Days)

25 . I: Saline
\ - Influenza
Q (U 20
8 g .
,g 2 15
.2 To
in in
as 10
< E
5 E 5
n.

o — — —

3 7 10 15 21
Time (Days)
15 *
* D saline

1:0 - Influenza
8
1:
2 10
O
.1:
.2
<
z
0
n.
a!

3 7 10 15 21
Time (Days)

Figure 8. Time-dependent proliferation of epithelial cells lining the main axial

airway following influenza infection.

E) Bronchoalveolar lavage fluid analysis
1. Protein
BALF-associated protein levels were significantly increased in samples collected
from mice infected with PR8 as compared with SAL instilled mice (Figure 9). Increased

protein levels were observed between 7 and 21 dpi with an apparent apex at 10 dpi.

2. Total and differential inflammatory cell counts

To further characterize the inflammatory cell milieu that was present in the
airways during these epithelial changes, differential cell counts were performed. There
was a significant rise in the total BALF-associated leukocytes (Figure 10A) in PR8
infected mice when compared to the respective SAL controls occurring as early as 3 dpi
and lasting through 15 dpi with an apparent apex at 10 dpi. The early innate immune
response was marked by significant increases in neutrophils (Figure 10B) by 3 dpi that
tapered by 10 dpi. Macrophages and other monocytic cells (Figure 10C) were
Significantly elevated between 7 and 15 dpi. The adaptive immune response was
characterized by marked increases in lymphocytes (Figure 10D) between 7 and 21 dpi
with peak numbers observed at 10 dpi. Eosinophils (Figure 10E) were also abundant

between 7 and 15 dpi.

3. Inflammatory chemokines and cytokines
With ongoing inflammation there are a host of chemokines and cytokines released
by activated leukocytes. To characterize the pro-inflammatory chemical mediators

retrieved in BALF, a mouse-specific cytometric bead array analysis of the inflammatory

54

   

 

 

1500' I: Saline
- Influenza

r_.1200-
%

3 900‘
C

39'; 600-
2

“- 300-

0 1"
3 7 1O 15 21
Time (Days)

Figure 9. Time-dependent detection of total protein in bronchoalveolar lavage fluid
following influenza infection. Effects of influenza instillation on total protein detected in
BALF supematants. Mice were instilled with PR8 or SAL and sacrificed 3, 7, 10, 15, and
21 dpi and concentrations of total protein in BALF were determined as described in
Materials and Methods. Data is expressed as mean i SEM. * = Significantly different

from respective control instilled with SAL.

55

Figure 10. Time-dependent recruitment of leukocytes to the pulmonary airways
following influenza infection. Effects of influenza instillation on total cells (A),
neutrophil (B), macrophage (C), lymphocyte (D) and eosinophil (E) retrieved in
bronchoalveolar lavage fluid (BALF). Mice were instilled with PR8 or SAL and
sacrificed 3, 7, 10, 15, and 21 dpi and inflammatory cells were enumerated in BALF as
described in Materials and Methods. Data is expressed as mean i SEM. * = significantly

different from respective control instilled with SAL.

56

Total number of cells I
ml (x1o‘)

 

 
   
 

Number of neutrophils /
ml (x103)

   

 

[:1 Saline
- lnfluenm

 

3 7 10 15 21
Time (Days)
\ D Saline 3 so I: Saline
g - Influenza g. - Influenza
C? t a g A t
g g 30 E-vo
,5 5 n 2 E
_ ~— _ 204
.. E 3 E
.8 1 .8 10‘ a *
E e . -
z _ __ _ _ _ g - _ __ __ _ _
3 7 10 15 21 3 7 10 15 21
Time (Days) Time (Days)

D Saline
- lnfluenm

   

Number of eosinophils /
ml (x103)

Time (Days)

Figure 10. Time-dependent recruitment of leukocytes to the pulmonary airways

following influenza infection.

57

cytokines TNF-a, IFN-y, lL-6, MCP-l, 1L-10, and IL-12p70 was employed. The BALF-
associated concentrations of TNF-or (Figure 11A), IFN-y (Figure 11B), IL-6 (Figure
11C), and MCP-l (Figure 11D), were significantly elevated in influenza infected mice
when compared to SAL controls at 7 dpi. In addition, concentrations of IL-10 (Figure
11E) were significantly decreased in PR8 infected mice as compared to SAL controls at
21 dpi. Levels of TNF-a and IL-6 remained Significantly elevated through 10 dpi with
similar trends observed with MCP-l. There were no changes in the BALF-associated

concentrations reported for IL-l2p70 (Figure 11F) with either PR8 or SAL instillation.

4. T;;2 cytokines IL-4, IL-5, IL-9 and IL-13

The TH2 cytokines IL-4 (Figure 12A), IL-5 (Figure 128), IL-9 (Figure 12C) and
IL-13 (Figure 12D) have been implicated in the development of MCM. By employing
cytometric bead array kits and flex sets for these cytokines, we observed marked
increases in the levels of IL-5 detected at 7 dpi in the BALF of PR8-infected mice as
compared to SAL instilled mice. Furthermore, there were significant, albeit mild
increases in the BALF levels of IL-4 and IL-9 detected at 7 dpi in PR8-infected mice as
compared to SAL instilled mice. The concentration of IL-13 in BALF was low in all
treatment groups with no detectable differences observed between PR8 and SAL instilled

mice.

5. Elastase

In addition to factors derived from lymphocytes, neutrophil-derived elastase has

also been implicated as a factor known to induce MCM. In the current study, neutrophil-

58

Figure 11. Secretion of chemokines and cytokines into the airways in response to
influenza infection. Effects of influenza instillation on TNF-a (A), IFN-y (B), IL-6 (C),
MCP-l (D), IL-10 (E), and IL-12p70 (F) retrieved in bronchoalveolar lavage fluid
(BALF). Mice were instilled with PR8 or SAL and sacrificed 3, 7, 10, 15, and 21 dpi and
inflammatory chemokines and cytokines were enumerated in BALF by flow cytometry as
described in Materials and Methods. Data is expressed as mean i SEM. * = significantly

different from respective control instilled with SAL.

59

300 D Saline

 

   

E g t - Influenza
e .. i
E g 100
3 a ‘
e V ‘
it 5
E L'-
3 7 10 15 21

Time (Days) Time (Days)

250 a D Saline A 400 D Saline
if 200 - Influenza I: 350 * - Influenza
A
< 150 s g
e 100 E 200
E 15 * E
g 10 T.’ 5°
3 E.) 25

2 0
3 7 10 15 21 3 7 10 15 21
Time (Days) Time (Days)
A 1°° D Saline G: 1° I: Saline
LL - Influenza 2’ - Influenza
2' ‘° 0
m _
_ .0 E
E 8
8 40 * 5’
[x
2. .. a
_J ‘-
— =1'
3 7 10 15 21 3 7 10 15 21
Time (Days) Time (Days)

Figure 11. Secretion of chemokines and cytokines into the airways in response to

influenza infection.

60

[:3 Same
- Influenza

’i

 

lL-4(pglmlBALF)
'i’ '1’ f 1'
It

 

3 7 10 15 21
Time (Days)

C3 Saline
- Influenza

lL-9 (pg/ml BALF)

asrr‘
(a)
I

 

 

7 10 15 21
Time (Days)

IL-5 (pg/ml BALF)

IL-13 (pg/ml BALF)

 

 

 

3 7 1O 15 21

 

Time (Days)

1 :3 Saline
in - Influenza
g...

4.1
w

Time (Days)

Figure 12. Detection of T32 cytokines secreted into the airways in response to

influenza infection. Effects of influenza instillation on the TH2 cytokines IL-4 (A), IL-5
(B), IL-9 (C), and IL-13 (D) retrieved in bronchoalveolar lavage fluid (BALF). Mice
were instilled with PR8 or SAL and sacrificed 3, 7, 10, 15, and 21 dpi and TH2 cytokines
were enumerated in BALF by flow cytometry as described in Materials and Methods.

Data is expressed as mean :t SEM. * = significantly different from respective control

instilled with SAL. N.D. = not detected.

61

derived elastase (Figure 13) was significantly elevated between 7 and 15 dpi in PR8

infected mice as compared to SAL control mice.

III. Modulation of airway responses to influenza A/PR/8/34 by delta-9-
tetrahydrocannabinol in C57BL/6 mice

A) A9-THC increases whole lung H1 mRNA following PR8 challenge

H1 mRNA levels in the lungs of mice challenged with PR8 in the absence of A9-
THC treatment were Significantly elevated at 7 dpi when compared to SAL control mice
(Figure 14). Levels of H1 mRNA detected in the lungs of mice challenged with PR8 and
treated with A9-THC at a dose of 25 mg/kg were also mildly attenuated when compared
to mice challenged with PR8 alone. The levels of H1 mRNA increased with increasing
doses of Ag-THC and were significantly elevated in mice administered 75 mg/kg A9-THC

when compared to PR8 challenged mice in the absence of A9-THC treatment.

B) A9-THC does not affect total protein levels in BALF

As a measure of alveolar/capillary membrane integrity, total protein in BALF was
assayed. There were marked increases in BALF-associated total protein observed in mice
challenged with PR8 at 7 dpi when compared to SAL control mice. However, there were
no differences observed in total BALF-associated protein recovered in PR8 challenged

mice treated with any dose of A9-THC (Figure 15).

C) A9-THC decreases leukocyte populations retrieved in BALF.

Consistent with an immune response to viral challenge, the total number of

62

 

10' * E Saline
- Influenza
s: 3' * *
<
2
2. 6"
0
fl,
.9 4'
In
L“
Ill 2.
o. — — — —
3 7 1O 15 21
Time (Days)

Figure 13. Secretion of neutrophil-derived elastase into the airways in response to
influenza infection. Effects of influenza instillation on neutrophil elastase detected in
BALF supematants. Mice were instilled with PR8 or SAL and sacrificed 3, 7, 10, 15, and
21 dpi and neutrophil elastase in BALF were determined as described in Materials and
Methods. Data is expressed as mean 1 SEM. * = significantly different from respective

control instilled with SAL.

63

7 days post infection

 

 

 

 

8,1000- :3 Saline
g Influenza
if; 800- #
E
Q 600-
,_
'2 400- *
.3
2
g 200-
(I
e, . a m
V I I I I I
I 0 0 25 50 75

 

Ag-THC (mg/kg)

Figure 14. A9-THC treatment enhances hemagluttinin 1 mRNA levels. Effects of A9-
THC on Hemagluttinin 1 mRNA levels from influenza challenge in whole lung
homogenates at 7 dpi in mice treated with SAL or Ag-THC (0, 25, 50, or 75 mg/kg) with
PR8. Data is expressed as mean i SEM; * = significantly different from respective

control instilled with SAL. # = significantly different from corn oil with PR8 challenge.

7 days post infection

30007 E Saline
Influenza
25001

 

 

2000-

 

1500-

Proteln (pig/ml)

S
‘3

 

 

 

 

 

 

\\\\\\\\\‘l
-k\\\\\\\\\i-I

 

 

<3

 

awn .

I I
0

25 50 75
A9-THC (mg/kg)

Figure 15. A9-THC does not alter total protein levels in BALF. Effects of Ag-THC on
total protein detected in BALF supernatants from mice challenged with influenza. Total
protein in BALF was determined for mice treated with SAL or Ag-THC (0, 25, 50, or 75
mg/kg) with PR8 and sacrificed at 7 dpi. Data is expressed as mean i SEM; * —

Significantly different from respective control instilled with SAL.

65

leukocytes retrieved in BALF was Significantly elevated in PR8 infected mice at 7 dpi
(Table 2). There was a trend toward dose-dependent effects of A9-THC treatment on the
total number of BALF-associated leukocytes. Treatment of mice with A9-THC at all dose
levels led to significant reductions in the number of BALF-associated lymphocytes.
Additionally, treatment of mice with A9-THC at doses of 25 mg/kg and 50 mg/kg led to a

reduction in the number of macrophages retrieved in BALF.

D) A9-THC decreases CD4+ and CD8+ T cells in BALF.

Since treatment of PR8 infected mice with A9-THC led to decreases in the number
of lymphocytes, we evaluated differences in T cell subsets by flow cytometry. The
absolute values of BALF-associated CD4+ and CD8+ T cells were determined (Table 3).
Treatment of PR8 infected mice with Ag-THC led to significant decreases in the number
of CD8+ T cells at all dose levels with respect to mice challenged with PR8 alone. A
similar effect on the number of CD4+ T cells was observed in PR infected mice
administered 50 and 75 mg/kg A9-THC as compared to mice challenged with PR8 in the

absence of A9-THC treatment.

E) A9-THC modestly affects chemokines and cytokines retrieved in BALF.

Inflammatory chemokines and cytokines secreted into the airways were measured
as an indicator of immune cell function in response to PR8 challenge. Mice challenged
with PR8 alone exhibited increased BALF concentrations of TNF-a , IFN-y, IL-6, MCP-
1, and IL-10 at 7 dpi as compared to the SAL control (Table 4). In PR8 infected mice

treated with A9-THC, an increase in MCP-l at a dose of 50 mg/kg A9-THC was observed,

66

 

[gal C0 217- THC 219- THC A”. THC

 

 

 

 

 

pe 25mg/kg 50mg/kg 75mg/kg
SAL PR8 PR8 PR8 PR8
Total 3.1:1.0 68.2:12.7 * 34.3:9.1 31.9:6.7 92:12.5
(1:10“) p = 0.020 = 0.170 r; = 0.058 E = 0.501
(4) 4)

 

 

10 ) p= 0.006 = 0.005 1p = 0.118

(4)
F:utrophils 2.9:2.0 1552.5:386. 9 E5)1.6:324.3 F586.3:412.5 2757.0:829.5
*

Fiaerophages 2.9:0.9 18.8:3.6 * F031. .3 # t.6i1.0 # 13.3:3.4

@;

 

 

x102) 0. 391 = 0.961 '1’ = 0.210

4) = 0.010 4

 

 

 

 

 

 

ymsphocytes 1.1:0.2 334.0:63.4 * 178. 8:50. 8 # 09:20.7 # 178.2:41.2 #
x10) p = 0.002 =0 019 = 0.004 1p = 0.047
(4)
osinophils 0 46.41163 * 3). 1:3. 0 33:21.3 48.617.86
x102) = 0.041 = 0.436 = 0.298 I = 0.654
(4) 4)

 

Table 2. Total and differential cell counts from BALF. The effects of A9-THC on the
recruitment of inflammatory cells to the pulmonary airways of mice challenged with
influenza. Mice were treated with A9-THC (25, 50, or 75 mg/kg) or its corn oil vehicle
for 5 consecutive days surrounding the intranasal instillation of influenza virus A/PR/8 or
its vehicle SAL. Mice were euthanized on day 7 post infection. BALF was collected by
flushing the lungs with 2 ml of sterile SAL. Total leukocyte counts were enumerated by
hemacytometer. Differential cell counts were assessed by counting 200 cells from
cytospins of the BALF stained with Diff-quick. Data is expressed as mean i SEM; n = 5
except where noted by (n); * = significantly different from respective control instilled

with SAL. # = significantly different from corn oil with PR8 challenge.

67

 

 

 

 

 

 

 

 

 

T-cell to A’— THC A’— THC ’- THC
Itype 25mg/kg 50mg/kg 75mg4kg
SAL PR8 PR8 PR8 PR8
CD4+ 154.6:25.4 883.7:96.8 * 38.2:77.4 304.4:15.5 # 475.4:64.0 #
(x104) p < 0.001 F = 0.626 < 0.001 < 0.001
(4) lo I0
CD8+ 66.7:265 4360.5:133.4 261.9:76.2 483.4:52.1 460.3:74.0 #
(x104) I: * E E F < 0.001
4) in < 0.001 < 0.001 < 0.001

 

 

 

Table 3. CD4+ and CD8+ T cell populations observed in BALF by flow cytometry.
The effects of A9-THC on lymphocyte populations of CD4+ and CD8+ T cells recruited to
the airways of mice challenged with PR8. Mice were treated with A9-THC (25, 50, or 75
mg/kg) or its corn oil vehicle for 5 consecutive days surrounding the intranasal
instillation of influenza virus A/PR/8 or its vehicle SAL. Mice were euthanized on day 7
post infection. BALF was collected by flushing the lungs with 2 ml of sterile SAL. CD4+
and CD8+ T cells in BALF were quantified by flow cytometric analysis. Data is
expressed as mean 3: SEM; n = 5 except where noted by (n); * = significantly different

from respective control instilled with SAL. # = significantly different from corn oil with

PR8 challenge.

68

 

 

 

 

 

 

 

 

 

 

 

 

 

hemokine/ Ico AT-THC AZTHC AT- THC
ytokine 25mg/kg 50mg/kg 75mg/kg
SAL PR8 PR8 PR8 PR8
IIL-6 09:05 497.5:862 * 32.711207 9549:2724 1120.0i168.9
(4) p= 0 001 = )0. 830 lp= 0.125 ip = 0.054
TNF-a. 6.7:4.7 145. 3+9. 9 * 129. 3+2. 9 145. 3+28. 0 155.8:262
(4)t14<)0. 001 k; 0.663 1p= 0.732 r= 0.977
FMCP-l 7.411. 1 1643. 0+296. 2 308. 0+70. 0 3301. 0+728. 4 5239017900
(4) * = )0 589 = 0.143
= 0.013 E4: 0.044
FN-y 1.5:0. 1 E4:11.0+263. 2 E2801+160 2 F415. .0+405. 0 l300.0_-L-489.4
(4) = 0.372 0.893 Ip = 0.815
0.048 4)
[IL-10 46.212.91.51 0:52. 0 L123. .0+22. 4 4. 2+11 .3 43.8:223
(4) =0 119 0.477 (4) =0 067 p = 0.100
L-12p70 35:04 .08+0. 6 .3+0. 7 .0+0. 4 5:1.2
(3);: 0.051 E= 0.884 F= 0.922 F= 0.855
4)

 

 

 

 

Table 4. Inflammatory cytokines quantified in BALF by cytometric bead array. The
effects of A9-Tl-IC on soluble mediators released by immune and epithelial cells in the
pulmonary airways during an inflammatory response to PR8 challenge. Mice were treated
with A9-THC (25, 50, or 75 mg/kg) or its corn oil vehicle for 5 consecutive days
surrounding the intranasal instillation of influenza virus A/PR/8 or its vehicle SAL. Mice
were euthanized on day 7 post infection. BALF was collected by flushing the lungs with
2 ml of sterile SAL. Concentrations of inflammatory cytokines in BALF were
enumerated by cytometric bead array analysis on the flow cytometer. Data is expressed as
mean i SEM; n = 5 except where noted by (n); * = significantly different from respective

control instilled with SAL. # = significantly different from corn oil with PR8 challenge.

69

Treatment of PR8 infected mice with Ag-THC at any dose, however, did not

significantly alter the BALF concentrations of TNF-a, IFN-y, IL-6, IL-10 or IL-12p70

when compared to mice infected with PR8 alone.

F) A9-THC treatment reduces inflammation scores for mouse lung sections.

The magnitude and severity of inflammation observed in histological sections of
lung isolated from the right and left lobes were independently scored (0 to 3, with O = no
inflammation and 3 = severe inflammation) and compared between treatment groups at 7
dpi (Figure 16A) and 10 dpi (Figure 16B). There was no inflammation observed in the
lungs of mice intranasally instilled with SAL. In contrast, there was a marked increase in
the inflammation score noted for lung sections obtained from mice challenged with PR8
alone at both 7 and 10 dpi, representing a moderate to severe inflammatory response.
There was a trend toward decreased inflammation scores for sections obtained from A9-
THC treated mice challenged with PR8 at 7 dpi. The inflammation scores for sections
obtained from Ag-THC treated mice challenged with PR8 were, however, significantly

attenuated at 10 dpi, representing mild to moderate levels of inflammation.

G) Effect of A9-THC on the observed pulmonary histopathology to PR8.

Exposure of mice to the corn oil vehicle or A9-THC alone did not result in
significant histologic changes within the control mice (Figure 17A). Infection of mice
with influenza induced a significant cellular and inflammatory reaction 7 dpi in all lung
regions examined. The inflammatory infiltrate was centered upon the bronchiolo-

alveolar duct junction, and extended out into the surrounding alveolar parenchyma. The

70

Figure 16. A9-THC decreases inflammation scores in a time-dependent manner.
Effects of A9-THC on the inflammatory response gathered within the subepithelial
interstitium and alveolar parenchyma following influenza challenge. Inflammation scores
were recorded from lung sections taken at 7 dpi (A) and 10 dpi (B). Scores were
tabulated as discussed in Materials and Methods. Data is expressed as mean i SEM; N.D.
= not detectable. * = significantly different from respective control instilled with SAL. #
= significantly different from corn oil with PR8 challenge.

fl
sum.
t
O
O.
y
d
7

m
6

af

3.

V///////////An
/7///////%6
///////////////.o

A9-rHc (mg/kg)

 

 

N.-o

 

 

“ d
3

2 1
200m coast—bacc—

10 da ays post infection

E..///////////..,.
...////////m
..////////.2.

.///////////////O

      
 

119-THC (mg/kg)

 

 

 

 

:BV

0.60m cow—«Epcot...

Figure 17. Examination of the effects of A9-THC on the inflammatory response to
influenza challenge by histopathology. Effects of A9-THC on the inflammatory
response observed at generation 5 of the main axial airway on day 10 post infection. Mice
were treated with Ag-THC (25, 50, or 75 mg/kg) or its corn oil vehicle for 5 consecutive
days surrounding the intranasal instillation of influenza virus A/PR/8 or its vehicle SAL.
Mice were euthanized on day 10 post infection. Lungs were fixed with 10% neutral-
buffered formalin for 24 hours, then sectioned and stained with hematoxylin and eosin
(H&E). Light photomicrographs were taken for lung sections at generation 5 of the main
axial airway (MAA) to include the bronchiolar artery (a) and alveolar parenchyma (p). A
= lung section from a mouse receiving corn oil gavage surrounding a SAL instillation.
There was no evidence of inflammation in the perivascular/peribronchiolar submucosal
compartment (sm) or alveolar parenchyma. B = lung section from a mouse receiving corn
oil gavage surrounding an influenza instillation. There was severe inflammation of the
perivascular/peribronchiolar submucosal compartment with marked extension into the
alveolar parenchyma. C = lung section from a mouse receiving THC (75 mg/kg) gavage
surrounding an influenza instillation. There was severe inflammation of the
perivascular/peribronchiolar submucosal compartment with modest extension into the
alveolar parenchyma. Bar = 100 microns. Images in this dissertation are presented in

color.

73

                

Dc .1 . a l U .(1‘.

a Y... .
ff .0 .n ..., Lg“: ‘ rs..fls
a an ...... .
. .. ... 41.4.6-0... ..
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-THC on the inflammatory response to

Figure 17. Examination of the effects of A9

histopathology.

influenza challenge by

74

inflammatory cells were a mix of primarily lymphocytes and neutrophils, with fewer
macrophages and plasma cells. The lymphocytic and neutrophilic population often filled
the alveoli, and there was moderate alveolar interstitial infiltration with similar
inflammatory cells. The lymphocytes often formed well-organized perivascular and

peribronchiolar aggregates. Acute epithelial necrosis was present in small numbers of
bronchioles, and the remaining epithelial cells were moderately attenuated. At 10 dpi
with influenza the inflammation was more severe, and often obscured the alveolar
parenchyma (Figure 17B). The inflammatory cells 10 dpi were primarily lymphocytes,
with smaller numbers of neutrophils. The bronchiolar epithelium 10 dpi was moderately
hyperplastic and hypertrophied, and there were scattered foci of alveolar
bronchiolarization (extension of bronchiolar epithelium into the adjacent alveolar
spaces). PR8-infected mice treated with Ag-THC resulted in no observed decreases in
inflammation 7 dpi at each A9-THC dose. At 10 dpi, exposure of PR8-infected mice with
all dose levels of Ag-THC resulted in a mild to moderate decrease in histologically
apparent inflammation within the lungs (Figure 17C). The inflammation within the mice
was not unifome distributed throughout all lung regions, as found in the control PR8-
infected mice. The decreases in inflammation included both decreases in inflammatory
cell numbers, as well as extent of distribution within the tissue. The bronchiolar

epithelial changes noted above were still present 10 dpi in the A9-THC co-exposed mice.
H) Caspase—3 mRNA expression levels and G5 numeric cell densities

CAS-3 is a cellular biochemical marker of committed activation of signaling

pathways that lead to cell death by apoptosis. In the current study, there were markedly

75

higher CAS-3 mRNA levels observed at 7 dpi in total lung homogenates from mice
challenged with PR8 alone as compared to mice instilled with SAL alone. The increase in
CAS-3 mRNA suggests an increased commitment to apoptotic cell death by cells present
in the lung in response to PR8 infection (Figure 18A). Ag-THC treated mice infected with
PR8 exhibited a trend toward increasing levels of CAS-3 mRNA with increasing doses of
Ag-THC when compared to mice infected with PR8 alone. In addition,
immunohistochemical staining for CAS-3 revealed marked increases in the number of
CAS-3 positive cells in lung sections obtained from mice infected with PR8 alone at 7
dpi (Figure 18B). There was a marked decrease (p = 0.056) in CAS-3 immunoreactive
epithelial cells at 7 dpi in PR8 infected mice treated with 25 mg/kg A9-THC when
compared to mice challenged with PR8 alone (Figure 18B). At 10 dpi, there were no
significant differences observed in the numeric cell densities for CAS-3 with any of the

treatments.

I) MUC5AC mRNA expression levels and G5 numeric cell densities

Increases in M UC5AC gene transcription might be an early indicator of increased
mucin production and possibly MCM in the bronchiolar epithelium. In the current study,
the levels of M UC5AC mRNA were increased in mice challenged with PR8 alone when
compared to mice instilled with SAL alone (Figure 19A). In Ag-THC treated mice
challenged with PR8, a dose of 25 mg/kg Ag-THC led to a four-fold increase in MUC5AC
mRNA levels as compared to mice infected with PR8 alone. In addition to changes in
MUC5AC gene expression, there were marked increases in the number of epithelial cells

staining for alcian blue (acidic mucosubstances) in lung sections obtained from mice

76

Figure 18. A9-THC has no effect on Caspase-3 mRNA levels and epithelial numeric
cell densities. The effects of Ag-THC on apoptotic cell death in response to influenza
challenge were measured by the expression of CAS-3 mRNA (A) and
immunohistochemical staining of CAS-3 in the epithelium lining the main axial airway at
generation 5 of the left lung lobe at 7 dpi (B) and 10 dpi (C). Mice were treated with SAL
or Ag-THC (O, 25, 50, or 75 mg/kg) with PR8. Data is expressed as mean :t SEM; N.D. =
not detectable. * = significantly different from respective control instilled with SAL.

77

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cell densities.

78

Figure 19. Ag-THC increases MUC5AC mRNA levels and decreases epithelial
numeric cell densities for acidic mucosubstances. The effects of Ag-THC on the
development of mucous cell metaplasia in the epithelium lining the main axial airway
following influenza challenge and subsequent inflammatory cell responses. Expression
levels of MUC5AC mRNA (A) and G5 main axial airway labeling indexes of alcian blue
were determined at 7 dpi (B) and 10 dpi for mice treated with SAL or Ag-THC (O, 25, 50,
or 75 mg/kg) with PR8. Data is expressed as mean i SEM; * = significantly different
from respective control instilled with SAL. # = significantly different from corn oil with

PR8 challenge.

79

7 days post infection

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10 days post infection
E Saline
lnfluenza

25

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Alcian blue positive epithelial cells I
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A9-THC (mg/kg)
Figure 19. Ag-THC increases MUC5AC mRNA levels and decreases epithelial

numeric cell densities for acidic mucosubstances.

80

infected with PR8 alone at 10 dpi but not at 7 dpi (Figures 19B and C). Ag-THC
treatment of PR8 infected mice did not affect the number of alcian blue stained epithelial
cells observed along the main axial airway at 7 dpi (Figure 198), but did attenuate the
number of alcian blue-positive cells observed at 10 dpi in mice receiving 75 mg/kg A9-

THC (Figure 19C) when compared to mice challenged with PR8 alone.

.1) A9-THC modestly enhances neutrophil-derived elastase levels in BALF

Neutrophil-derived elastase is another soluble mediator secreted into the airways
during ongoing inflammation that has been shown to have an influence on the
development of MCM. Neutrophil-derived elastase was not detectable in BALF from
SAL instilled mice (Figure 20). However, BALF-associated elastase was markedly
increased in mice infected with PR8 alone. Ag-THC treated mice infected with PR8
exhibited a modest increase in elastase levels at a dose of 75 mg/kg Ag-THC when

compared to mice challenged with PR8 alone.

IV. Pulmonary airway responses to influenza A/PR/8/34 in CBI'I'lCBz’l' mice
exposed to A9-tetrahydrocannabinol: An examination for the role of cannabinoid
receptors

A) Qualitative health assessment of CBf’VCBf” and wild type mice
challenged with PR.

CBl‘l’lCBg'l' and wild type mice treated with corn oil or Ag-THC and intranasally
instilled with SAL were normal in appearance and activity level. Infection with PR8 in

the presence or absence of A9-THC treatment, however, led to marked differences in the

81

7 days post infection

Saline

25- El
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Figure 20. Ao-THC modestly increases neutrophil-derived elastase levels secreted in
the pulmonary airways. Effects of Ag-THC on the secretion of elastase by neutrophils in
response to influenza infection within the pulmonary airways at 7 dpi. Neutrophil-derived
elastase in BALF was determined for mice treated with SAL or Ag-THC (0, 25, 50, or 75
mg/kg) with PR8. Data is expressed as mean 2: SEM; * = significantly different from
respective control instilled with SAL. N.D. = not detectable. # = significantly different

from corn oil with PR8 challenge.

82

gross appearance of CB1'/'/CB2'/’ and wild type mice. Specifically, CB1'/'/CB2'/' mice were
notably more gaunt in comparison to wild type mice, suggesting that the mice were
dehydrated. In addition, the CBl'/'/CB2'/' mice were lethargic and displayed unkempt fur.
In contrast, wild type mice infected with PR8 in the presence or absence of Ag-THC
treatment maintained normal grooming habits and exhibited similar activity levels as the
SAL instilled controls. Upon gross examination of the lungs, CBl'/'/CB2'/' mice had
extensive hemorrhaging across all lung lobes. Wild type mice also displayed evidence of

hemorrhaging. however, to a much lesser extent than CB|'/'/CB2'/' mice.

B) The viral load of PR8 in the pulmonary airways of CB;"’/CB2"' and wild
type mice measured 7 days after challenge.

The levels of mRNA for the highly antigenic viral surface protein, hemagglutinin
1, were assessed by quantitative real time PCR. For both CB1'/'/CB2'/' and wild type mice,
viral H1 expression was markedly elevated above SAL controls at 7 dpi in the lungs of all
mice challenged with PR8 (Figure 21). Infection of A9-THC treated cert/€132"- mice
with PR8 resulted in an increase (p = 0.056) in H1 mRNA levels when compared to mice
instilled with PR8 alone. In A9-THC treated wild type mice infected with PR8, there was
a significant increase in viral H] mRNA levels when compared to wild type mice
instilled with PR8 alone. The overall level of expression of H1 mRNA, though, was

greatly reduced in CB.'/'/CB2'/' mice when compared to wild type mice.

C) A9-THC affects vascular permeability induced by PR8 infection of the

airways in CBf’VCBf’“ and wild type mice.

83

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Figure 21. Effects of A9-THC treatment on the expression levels of H1 mRNA in
CBl‘/‘/CB2'/' and wild type mice. The effects of Ag-THC on Hemagglutinin 1 mRNA
levels in lungs from CB1'/'/CB2'/' and wild type mice challenged with PR8. Mice were
treated with Ag-THC (75 mg/kg) or its corn oil vehicle for 5 consecutive days
surrounding the intranasal instillation of influenza virus A/PR/8 or its vehicle SAL. Mice
were euthanized on day 7 post infection. Whole lungs were immersed in TRI reagent and
homogenized prior to the isolation of total RNA. The mRNA levels for Hemagglutinin 1
were determined by real-time PCR with 18S utilized as an internal loading control. The
fold-change in gene expression is normalized to mice instilled with SAL. Data is
expressed as mean i SEM; * = significantly different from respective control instilled
with SAL. # = significantly different from respective control gavaged with corn oil. + =

significantly different from respective group in wild type mice.

84

The detection of increased levels of total BALF-associated protein is indicative of
increased vascular, permeability at the alveolar/capillary interface during the
inflammatory response to PR8. The total BALF-associated protein levels in PR8 infected
CB1'/'/CB2'/' and wild type mice, in the absence of A9-THC treatment, were 2-fold greater
than SAL controls (Figure 22). A9-THC treated CB1'/'/CB2'/' mice infected with PR8
exhibited a 33% increase in total protein when compared to CB1'/'/CB2'/' mice challenged
with PR8 alone. Conversely, Ag-THC treated wild type mice infected with PR8 had a
50% decrease in total protein when compared to wild type mice challenged with PR8
alone. When comparing CBI"'/CB2'" and wild type mice, the amount of total protein
detected in mice treated with Ag-THC and infected with PR8 was 2-fold greater in CBl'l'
/CB2'/' mice than amounts of total protein observed in wild type mice receiving the same
treatment.

D) CBl'l'lCBz'I' mice treated with A9-THC exhibit a distinctly different
composition of leukocytes recruited to the airways in response to PR8 infection
when compared to wild type.

Primary influenza infection elicits an inflammatory response consisting of a
mixed population of leukocytes that infiltrate the pulmonary airways. In particular, there
is a marked influx of neutrophils and lymphocytes into the airways with monocytes and
eosinophils representing a smaller portion of the total population of BALF-associated
leukocytes. In the current study, the total number of BALF-associated leukocytes was 3-
fold greater in PR8-infected wild type mice and 4-fold greater in CB 14 '/CB2'/ ' mice when
compared to their respective non-infected controls (Figure 23A). A9-THC treated mice

infected with PR8 exhibited a 2-fold increase above SAL instilled controls in both

85

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Figure 22. Effects of Ag-THC treatment on total protein in BALF in CBI'I'ICBz'I' and
wild type mice. The effects of Ag-THC on total protein detected in BALF supematants
from CB."l‘/CB2'/' and wild type mice challenged with PR8. Mice were treated with A9-
THC (75 mg/kg) or its corn oil vehicle for 5 consecutive days surrounding the intranasal
instillation of influenza virus A/PR/8 or its vehicle SAL. Mice were euthanized on day 7
post infection. BALF was collected by flushing the lungs with 2 ml of sterile SAL. Total
protein was assessed for supematants from BALF that had been centrifuged to remove
cellular debris. Data is expressed as mean i SEM: * = significantly different from
respective control instilled with SAL. + = significantly different from respective group in

wild type mice.

86

CB1’/'/CBz'/' and wild type mice. However, the difference in total leukocytes retrieved in
the Ag—THC treated CB1'/'/CB2'/' mice infected with PR8 was significantly less than CBt'l'
/CB2'/' mice challenged with PR8 alone. To further assess differences in individual
leukocyte populations retrieved by lavage, differential cell counts were performed.
Neutrophils (Figure 23 B) were markedly increased in PR8-infected CBt'l'lCB2'/' and wild
type mice. Specifically, Ag-THC treated wild type mice infected with PR8 exhibited 2-
fold increases in neutrophils when compared to wild type mice challenged with PR8
alone. Conversely, there were marked decreases in the number of BALF-associated
neutrophils for PR8-infected CB1'/'/CBz'/' mice treated with Ag-THC when compared to
CBt'/'/CB2'/' mice challenged with PR8 alone. BALF-associated lymphocytes were
markedly increased (Figure 23C) with PR8 infection in both CBf/‘lCBz'l' and wild type
mice. Ag-THC treated cert/C133“ and wild type mice infected with PR8 exhibited
attenuation in the number of lymphocyte retrieved. There were no apparent changes in
the number of macrophages retrieved in BALF from PR8 challenged wild type mice. A
2-fold increase was observed, however, in the number of BALF-associated macrophages
from PR8-infected CB1'/’/CBz'/' mice that was attenuated in Ag-THC treated CB1'/'/CB2'/'
mice infected with PR8 (Figure 23D). Lastly, PR8-infection of CB1" ”/CB2’/ ' and wild type
mice resulted in an increase in the number of eosinophils retrieved in BALF (Figure
23E). A9-THC treated CBl'/'/CB2'/' and wild type mice infected with PR8 exhibited no

effect in the number of BALF-associated eosinophils counted.

87

Figure 23. Effects of A9-THC treatment on total and differential leukocyte counts in
BALF in CBf”/CB2"' and wild type mice. The effects of Ag-THC on leukocyte
recruitment to the pulmonary airways of CBt'/'/CB2'/' and wild type mice challenged with
PR8 were enumerated. Mice were treated with Ag-THC (75 mg/kg) or its corn oil vehicle
for 5 consecutive days surrounding the intranasal instillation of influenza virus A/PR/8 or
its vehicle SAL. Mice were euthanized on day 7 post infection. BALF was collected by
flushing the lungs with 2 ml of sterile SAL. Total leukocyte (A) counts were enumerated
by hemacytometer. Differential cell counts of neutrophils (B), lymphocytes (C),
macrophages (D), and eosinophils (E) were assessed by counting 200 cells from
cytospins of the BALF stained with Diff-quick. Data is expressed as mean :1: SEM; * =
significantly different from respective control instilled with SAL. # = significantly
different from respective control gavaged with corn oil. + = significantly different from

respective group in wild type mice.

88

   

   
   

 

 

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89

E) BALF-associated CD4+ and CD8+ T cell levels following PR8 challenge in CBf"
/CB;"' and wild type mice.

To evaluate the contribution of CD4+ and CD8+ T cells within the pool of BALF-
associated lymphocytes responding to PR8 challenge, the number of CD4+ T cells
(Figure 24A) and CD8+ T-cells (Figure 24B) were enumerated by flow cytometric
analysis. Wild type mice treated with either corn oil or Ag-THC exhibited modest levels
of BALF-associated CD44r T cells. Interestingly, background levels of CD4+ T cells were
greater in corn oil and A9-THC treated CB1'/'/CBZ'/' mice than in wild type mice with the
same treatments. As a result, there was no observed difference in the number of CD4+ T
cells retrieved in BALF between SAL instilled controls and CBt'/'/CB2'/' mice infected
with PR8 alone. Alternatively, Ag-THC treated CBt'/'/CB2’/' mice infected with PR8
exhibited a significant increase in CD4+ T cells above background. The increase in
BALF-associated CD4+ T cells in CB|'/'/CB2'/' mice treated with Ag-THC and infected
with PR8 was comparatively greater than the respective treatment group in wild type
mice. CD8+ T cells were not detected in BALF from SAL instilled controls in both CBf"
/CB2‘/' and wild type mice. However, there were marked increases in the number of
BALF-associated CD8+ T cells for both CB1'/'/CB2'/' and wild type mice challenged with
PR8. In addition, there was a trend toward decreased numbers of BALF-associated CD8+

T cells in A9-THC treated wild type mice infected with PR8.

90

Figure 24. Effects of A9-THC treatment on CD4+ and CD8+ T cells retrieved in
BALF in cert/cm" and wild type mice. The effects of Ag-THC on CD4+ T cell (A)
and CD8+ T cell counts (B) recruited to the airways of CBt'/'/CB2'/' and wild type mice
challenged with influenza. Mice were treated with Ag-THC (75 mg/kg) or its corn oil
vehicle for 5 consecutive days surrounding the intranasal instillation of influenza virus
A/PR/8 or its vehicle SAL. Mice were euthanized on day 7 post infection. BALF was
collected by flushing the lungs with 2 ml of sterile SAL. CD4+ and CD8+ T cells in
BALF were quantified by flow cytometric analysis. Data is expressed as mean + SEM; *
= significantly different from respective control instilled with SAL. # = significantly
different from respective control gavaged with corn oil. + = significantly different from

respective group in wild type mice. N.D.= not detected.

91

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BALF in CB."‘/CB;"' and wild type mice.

92

F) Cannabinoid receptor deficient mice exhibit unique differences in
epithelial and leukocytic chemokine and cytokine secretion in the pulmonary
airways.

One mechanism of cellular communication within a mixed population of
leukocytes and between infected epithelium and leukocytes is through secretion of
cytokines and chemokines. PR8-infected CB1'/'/CB2'/' mice exhibited marked increases in
the chemokine MCP-l (Figure 25A), and pro-inflammatory cytokines TNF-a (Figure
25B), IL-6 (Figure 25C), and IFN-y (Figure 25D) measured in BALF. Likewise, wild
type mice challenged with PR8 exhibited increased levels of BALF-associated MCP-l,
TNF-a, IL-6, and IFN-y. In addition, there was a modest enhancement of IL-10
concentrations (Figure 25E) following PR8 challenge in CB1'/'/CB2’/' and wild type mice.
There were no changes in BALF-associated IL-l2p70 in the presence or absence of A9-
THC treatment or PR8 challenge (Figure 25F) in either CB1’/'/CB2‘/' or wild type mice.
Ag-THC treated CBt'/'/CB2"' mice infected with PR8 exhibited an attenuation of BALF-
associated IFN-y and IL-10 concentrations. Conversely, the concentrations of chemokines
and cytokines detected in BALF in PR8-infected wild type mice were unaffected by A9-
THC treatment. There were, however, comparative differences between detectable levels
of BALF-associated TNF-a, and IFN-y (p = 0.061) in PR8-infected wild type and CB1'/’
/CB2'/' mice.

The BALF-associated TH2 cytokines IL-2 (Figure 26A), IL-4 (Figure 26B), and
IL-5 (Figure 26C) were also quantified by cytometric bead array. Of particular interest,
PR-8 infected CB1'/'/CB2'/' mice exhibited marked increases in concentrations of IL-5 that

were comparatively 2-fold less than concentrations observed in the cognate PR8-infected

93

Figure 25. Effects of A9-THC treatment on inflammatory chemokines and cytokines
in secreted into the pulmonary airways in CBI'I'ICBz'l' and wild type mice. The
effects of A9-THC on the release of soluble chemical mediators from leukocytes and
epithelium in response to PR8 challenge of the pulmonary airways of CB.'/'/CB2'/' and
wild type mice. Mice were treated with A9-THC (75 mg/kg) or its corn oil vehicle for 5
consecutive days surrounding the intranasal instillation of influenza virus A/PR/8 or its
vehicle SAL. Mice were euthanized on day 7 post infection. BALF was collected by
flushing the lungs with 2 ml of sterile SAL. Concentrations of the inflammatory
chemokine MCP-l (A) and cytokines TNF-a (B), IL-6 (C), IFN-g (D), IL-lO (E) and IL-

12p70 (F) were determined by cytometric bead array analysis. Data is expressed as mean

:t SEM; * — significantly different from respective control instilled with SAL. # =
significantly different from respective control gavaged with corn oil. + = significantly

different from respective group in wild type mice.

94

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in secreted into the pulmonary airways in CBt'l'lCBz'l' and wild type mice.

95

Figure 26. Effects of A9-THC treatment on TH2 cytokines secreted into the
pulmonary airways in CBl'I'lCBz'I' and wild type mice. The effects of A9-THC on the
concentrations of the TH2 cytokines IL-2 (A), IL-4 (B), and IL-5 (C) in BALF from CB1'/ '
/CB2'/’ and wild type mice challenged with PR8. Mice were treated with Ag-THC (75
mg/kg) or its corn oil vehicle for 5 consecutive days surrounding the intranasal
instillation of influenza virus A/PR/8 or its vehicle SAL. Mice were euthanized on day 7
post infection. BALF was collected by flushing the lungs with 2 ml of sterile SAL.
Cytometric bead array was performed on BALF to enumerate the TH2 cytokines. Data is
expressed as mean :1: SEM; * = significantly different from respective control instilled
with SAL. # = significantly different from respective control gavaged with corn oil. + =

significantly different from respective group in wild type mice.

96

lL-4 (pg/ml) lL-2 (pg/ml)

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Figure 26. Effects of A9-THC treatment on TH2 cytokines secreted into the
pulmonary airways in CB1'/'/CB2'/' and wild type mice.

97

wild type mice. The concentrations of lL-2 and IL-4 in BALF were unaffected by PR8
challenge in CB.'/'/CBz'/' mice. Ag—THC treated CB1'/'/CBz'/’ mice infected with PR8 had
modestly attenuated concentrations of BALF-associated IL-2, but exhibited no effect on
the concentrations of lL-4 or IL-5. PR8-infected wild type mice exhibited marked
increases in BALF IL-5 concentrations, subtle increases in the concentrations of IL-2,
and no detectable differences for IL-4 when compared to SAL instilled mice. A9-THC
treated wild type mice infected with PR8 had a modest attenuation of BALF IL-2

concentrations with no observed effect on either IL-4 or IL-5 concentrations.

G) Cytokines and chemokines detected in blood serum.

In CB 1'/'/CB2’/' mice, PR8 challenge alone resulted in marked increases in serum
associated MCP-l (Figure 27A), TNF-a (Figure 27B), lL-6 (Figure 27C), and IFN-
y (Figure 271)) with respect to SAL control. In wild type mice, PR8 challenge increased
circulating levels of the chemokine, MCP-l and the cytokine IFN-y with respect to SAL
instilled mice. In CB1'/'/CB2'/' there was no difference observed between SAL controls
and PR8 challenge for serum concentrations of IL-12p70 (Figure 27E). In wild type mice
there were modest, yet significant, increases observed in the concentrations of IL-12p70
with PR8 instillation as compared to SAL instillation alone in the presence or absence of
Ag-THC. CB1'/'/CB2'/' mice treated with Ag-THC had attenuated concentrations of MCP-l
and IFN-y with respect to PR8 challenge alone, whereas wild type mice treated with A9-
THC exhibited markedly enhanced circulating levels of IL-6 with respect to mice
challenged with PR8 alone. In addition, there were comparative differences observed

between CB1'/'/CB2'/' and wild type mice with PR8 challenge for the concentrations of

98

Figure 27. Effects of A9-THC treatment on inflammatory chemokines and cytokines
released into blood serum in CBI'I'ICBz'l' and wild type mice. The effects of A9-THC
on the dispersion of soluble chemical mediators from leukocytes and epithelium into the
circulation in response to PR8 challenge of the pulmonary airways of CB1'/'/CB2'/' and
wild type mice. Mice were treated with A9-THC (75 mg/kg) or its corn oil vehicle for 5
consecutive days surrounding the intranasal instillation of influenza virus A/PR/8 or its
vehicle SAL. Mice were euthanized on day 7 post infection. Serum was collected from
the abdominal aorta. Concentrations of the inflammatory chemokine MCP-l (A) and
cytokines TNF-a (B), IL-6 (C), IFN-g (D), and IL-12p70 (E) were determined by
cytometric bead array analysis. Data is expressed as mean i SEM; * = significantly
different from respective control instilled with SAL. # = significantly different from

respective control gavaged with corn oil. N.D.= not detected.

99

a Wild type

:1 Wild type
-. cert/cm"

- cert/ca."-

   
  

   
   
   

 

 

g ... a
3 a
‘_ 100 ‘j
o'. it
o
2 5
Com oil A9-THC Comoil Ag-THC Corn oil Ag-THC Comoil Ag—THC
Saline lnfluenza Saline lnfluenza

1: Wild type :1 Wild e
- CB,"'/CBZ‘"

- CB."‘/CBZ"‘

 

 

so

A 2 ‘°
E E
s 3 3°
3 e

— 10

Comoil Ag-THC Cornell AS-THC Comoil Aime Comoil ,9ch
Saline lnfluenza Saline lnfluenza
10 D Wild type

- cert/cs,"-

lL-12p70 (pg/ml)

Comoil Ag-THC Corn oil 119-THC
Saline Influenza

 

Figure 27. Effects of Ag-THC treatment on inflammatory chemokines and cytokines

released into blood serum in CBi'/'/CB{" and wild type mice.

100

MC P-l detected.

H) The absence of cannabinoid receptors CB; and CB; enhances the
observed pulmonary histopathology.

As previously reported by our laboratory, infection of mice with PR8 induces
significant pulmonary inflammation 7 dpi in wild-type mice (Figure 28A-D) (113).
Infection of the CBi'/'/CB2'/' mice with PR8 resulted in a similar inflammatory reaction
(Figure 28E). As we have reported, the inflammation consists of primarily lymphocytes
and neutrophils, with fewer macrophages and plasma cells centered upon the bronchiolo-
alveolar junction, and extending out into the surrounding parenchyma. Treatment of PR8
infected wild-type mice with A9-THC resulted in a significant decrease in inflammation 7
dpi compared to corn oil treated mice infected with PR8 (Figure 28D). These mice often
had no to few inflammatory cells present; the remaining inflammation, was comprised of
primarily lymphocytes and alveolar macrophages. In contrast, treatment of PR8 infected
CBt'/'/CB2'/' mice with A9—THC resulted in a vigorous inflammatory and cellular reaction
in the mice (Figure 28F). In these mice there were large numbers of mature lymphocytes
around the conducting airways and extending into the surrounding alveolar parenchyma
where they were admixed with fewer macrophages and neutrophils. The bronchiolar
epithelium was moderately hypertrophied. Treatment of wild type and CBi'/'/CB2'/' mice
with corn oil vehicle or Ag-THC alone did not result in significant histologic changes in

any of the lung sections examined.

101

Figure 28. Inflammatory response to PR8 in the proximal section of the left lung
lobe in CBl'l’lCBz'l' and wild type mice. Light photomicrographs of the hilar region of
the left lung lobe of wild type (WT) and CB1'/'/CB2"' (KO) mice sectioned at generation 5
along the main axial airway (maa). Panel (A): corn oil treated wild type mouse instilled
with saline (SAL) with no evidence of an inflammatory cell infiltrate (arrow) in the
peribronchiolar (maa)/perivascular (a) submucosa (sm) and no evidence of inflammation
in the alveolar parenchyma (p). Panel (B): A9-THC-treated wild type mouse instilled with
saline with marked inflammatory cell infiltrate of the submucosa and diffuse
inflammation within the alveolar airspace. Panel (C): corn oil treated wild type mouse
instilled with PR8 with marked inflammatory cell infiltrate in the peribronchiolar
/perivascular submucosa and alveolar airspace. Panel (D): Ag-THC-treated wild type
mouse instilled with PR8 with modest to no inflammatory cell infiltrate of the submucosa
and alveolar airspace. Panel (E): corn oil treated CBt’/'/CBz'" mouse instilled with PR8
with marked inflammatory cell infiltrate in the peribronchiolar /perivascular submucosa
and alveolar airspace. Panel (F): A9-THC-treated CB1'/'/CB2"' mouse instilled with PR8
with severe inflammation within the submucosa and alveolar airspace. Bar = 50 microns.

Images provided in this dissertation are in color.

102

9

Corn oil treated A -THC treated
A 1&5": ’9"! '3' 1"!” be"? B ~ .' 153%}: r'i‘l‘i-K,
. ' , _ \-v‘:"‘ ‘1‘ ' ' ’1': . I. {k

      

Figure 28. Inflammatory response to PR8 in the proximal section of the left lung

lobe from wild type mice.

103

I) A9-THC affects the magnitude of the inflammatory response to PR8 in wild type
and CB] and CB; deficient mice.

The magnitude and severity of inflammation observed in tissue sections isolated
from the left lung lobe were independently scored (0 to 3, with 0 = no inflammation and
3 = severe inflammation) and compared between treatment groups and wild type and
CBi'/’/CB2'/' mice at 7 dpi (Figures 29). There was no inflammation observed in the lungs
of wild type mice intranasally instilled with SAL. However, 2 out of 8 CBi'I'lCBz'I' mice
instilled with SAL exhibited modest evidence of ongoing inflammation. Indeed, the CB{
"/CBZ'I' mice were subjected to an extensive battery of serological screens prior to their
use and were found to be negative for all pathogens tested. Interestingly, there was a
marked and identical increase in the inflammation score noted for lungs in PR8-infected
wild type and CBi'/'/CB2'/' mice. Similar to previous findings by our laboratory (113), A9-
THC treated wild type mice challenged with PR8 exhibited a suppression of the
inflammatory response in the lung airways. In marked contrast, A9-THC treated CBf”
/CB2'/' mice infected with PR8 had a trend toward increased (p = 0.079) inflammation of

the pulmonary airways.

J) Effects of CB1 and CB; deficiency on the numeric cell densities of
apoptotic cells and metaplastic goblet cells.

Bronchiolar epithelial cell apoptosis in influenza-infected mice is directed by cell-
mediated immune responses to virally infected cells (114). The numeric cell density for
the apoptotic cell marker, CAS-3 (Figure 30) was quantified at 7 dpi in the epithelium

lining generation 5 of the main axial airway. CAS-3 numeric cell densities were not

104

3.0- 1: Wild type
25 - cefivcefi

2.0-

* +
1.5- * *
1.0-l #
0.5- lll
0.0- ND. i N.D. i

Corn oil A9-THC Corn oil All-THC

Inflammation score

 

 

 

Saline lnfluenza

Figure 29. Effects of A9-THC treatment on histopathology-based inflammation
scores in CBi'/'/CB2‘/' and wild type mice. Effects of Ag-THC on the inflammatory
response gathered within the subepithelial interstitium and alveolar parenchyma
following influenza challenge of the pulmonary airways in CB1'/'/CB2'/' and wild type
mice. Mice were treated with A9-THC (75 mg/kg) or its corn oil vehicle for 5 consecutive
days surrounding the intranasal instillation of influenza Virus A/PR/8 or its vehicle SAL.
Mice were euthanized on day 7 post infection. Lungs were fixed with 10% neutral-
buffered formalin for 24 hours, then sectioned and stained with hematoxylin and eosin
(H&E). Inflammation scores were recorded from lung sections taken on 7 dpi. Scores
were tabulated and averaged for the two sections taken from generations 5 and 11 of the
left lung lobe. Scores: 0 = no inflammation, l= mild, inflammatory cell infiltrate of the
perivascular/peribronchiolar compartment, 2 = moderate, inflammatory cell infiltrate of
the perivascular/peribronchiolar space with modest extension into the alveolar
parenchyma, and 3 = severe, inflammatory cell infiltrate of the
perivascular/peribronchiolar space with a greater magnitude of inflammatory foci found
in the alveolar parenchyma. Data is expressed as mean :h SEM; N.D. = not detectable. * =
significantly different from respective control instilled with SAL. + = significantly

different from respective group in wild type mice.

105

2.5- D Wild type
* - ce1"'/c324'
2.0-

1.5-
1.0-
0.5-
0,. N.D.N.D. N.D.N.D. N.D.

Corn oil Ag-THC Corn oil A°.THC

Caspase-3 positive cells I
mm basal lamina

 

 

 

 

Saline lnfluenza

Figure 30. Effects of A9-THC treatment on the numeric cell density of caspase-3
positive epithelial cells in CBl'I'lCBz'I' and wild type mice. The effects of Ag-THC on
apoptotic cell death in response to influenza challenge. CB1'/'/CB2"/' and wild type mice
were treated with Ag-THC (75 mg/kg) or its corn oil vehicle for 5 consecutive days
surrounding the intranasal instillation of influenza virus A/PR/8 or its vehicle SAL. Mice
were euthanized on day 7 and 10 post infection. Lungs were immersed in TRI reagent for
the isolation of total RNA or fixed with 10% neutral-buffered formalin for 24 hours, then
sectioned and stained by immunohistochemistry for Caspase-3. Numeric cell densities
were determined by enumerating caspase-3 positive nuclei per length of basal lamina.
Data is expressed as mean i SEM; * = significantly different from respective control
instilled with SAL. # = significantly different from respective control gavaged with corn

oil. + = significantly different from respective group in wild type mice.

106

distinguishably different between treatment groups in wild type mice. In PR8-infected
CB1'/'/CB2'/' mice, CAS-3 levels were modestly increased above SAL instilled controls,
but were no different than cell densities enumerated for CB1°/'/CB2’/' mice infected with
PR8 and treated with Aq-THC. During the recovery from viral infection a metaplastic
change occurs in the epithelium lining the bronchi that includes increased numbers of
mucus producing goblet cells (115). Consistent with our previous finding (113), there
was no difference in the numeric cell density of mucus production (Figure 31) quantified
at 7 dpi in wild type mice with any treatment. Rather interestingly, Ag-THC treated CBi‘/'
/CBz'/' mice in the presence or absence of PR8 infection, exhibited significant increases
in the numeric cell density for mucin-positive epithelial cells as compared to wild type

mice.

107

   
 

 

Mucin positive cells I
mm basal lamina

35- * + 1: Wild type
30- - cep’vcey'
25-

20- +

15-

10-

5 fli ii

0 7 —

Com oil Ail-THC Corn oil A9-THC

 

 

Saline Influenza

Figure 31. Effects of Ag-THC treatment on the numeric cell density of
mucosubstances in airway epithelial cells in CB.'/'/CBz'/' and wild type mice. The
effects of Ag-THC on the development of mucous cell metaplasia in the epithelium lining
the main axial airway following influenza challenge and subsequent inflammatory cell
responses. CBfL/CBZ‘X' and wild type mice were treated with Ag-THC (75 mg/kg) or its
corn oil vehicle for 5 consecutive days surrounding the intranasal instillation of influenza
virus A/PR/8 or its vehicle SAL. Mice were euthanized on days 7 and 10 post infection.
Lungs were fixed with 10% neutral-buffered formalin for 24 hours, then sectioned and
stained with alcian blue/periodic acid Schiff (neutral and acidic mucosubstances).
Numeric cell densities were determined by enumerating alcian blue positive secretory
vesicles in epithelial cells per length of basal lamina from stained sections. Data is
expressed as mean + SEM; * = significantly different from respective control instilled
with SAL. # = significantly different from respective control gavaged with corn oil. + =

significantly different from respective group in wild type mice.

108

DISCUSSION

The studies outlined in this dissertation were a part of an effort to evaluate the
effects of A9-THC and the role of the cannabinoid receptors CB1 and. CB2 on immune and

pulmonary epithelial cell responses in a murine model of host-resistance to influenza

virus A/PR/8/34 (Table 5).

1. Animals utilized in these studies

In the studies outlined in this dissertation, C57BL/6 mice were utilized. C57BL/6
mice do not exhibit a genetically determined preference to either a THl or TH2
predominant immune response to pathogens like other mouse strains (e.g. Al or BALB-C
mice). In addition, CB1/C82 receptor knockout mice were developed on a C57BL/6

background.

11. The method of delivery and the dose of PR utilized

In the studies outlined in this dissertation, a PR8 exposure paradigm was chosen
such that the mice were instilled with a low concentration (50 pfu) of PR8 to ensure the
survival of the mice for the duration of the kinetic study (21 days). In addition, PR8 was
intranasally instilled in a large volume of SAL (50 ill) to facilitate the delivery of PR8 to
the lower airways. This paradigm is unlike most studies, wherein immune responses to
influenza are examined within the first 6 — 10 days following a high concentration virus

exposure.

III. The method of delivery and dose of A9-THC utilized in these studies

There are two ma'or routes of ex osure to Ag-THC in humans, inhalation and oral
.l P

109

I1

 

Effect of corn oil on: Wild CB
Saline Saline
E

MCM
H&E
Inflammato cells
BALF Leuk
BALF C kines

Histoc

Effect of A -THC on: Wild T CB] /CBz
Saline Saline PR8

MCM -H-+ +
H&E --- .H.
Inflamma cells
BALF Leuk +
BALF C +
Histochemi +1—1-

 

Table 5. Summary of the effects of corn oil and A9-THC on BALF and
histochemistry measurements taken for epithelial and inflammatory cells in wild
type and CBt'l'lCBz'l' mice. Dashed line denotes no effect. A single plus denotes a mild
effect, a double plus indicates a modest effect, and a triple plus signifies a marked

response.

110

consumption. In the studies outlined in this dissertation, Ag-THC was administered orally
in corn oil for five consecutive days. The oral route of administration was selected due to
the fact that Ag-THC is a highly lipophilic molecule requiring a nonaqueous diluent for
drug delivery, which in itself has the likely potential for inducing irritation and damage to
the airways. In contrast, oral administration of Ag-THC in corn oil is well tolerated but
poorly absorbed from the gastrointestinal tract resulting in modest blood concentrations
of Ag-THC and its metabolites. Oral administration of 75 mg/kg A9-THC for 5
consecutive days resulted in a serum concentration of 66.2 ng/ml of the parent
compounds, 446.5 ng/ml of the 9-COOH metabolite and 10.5 ng/ml of the ll-OH
metabolite, four hours after the last Ag-THC dose. The levels of Ag-THC observed
systemically correlate well with a previous report by Azorlosa and coworkers (1992)
(116) in which peak human plasma levels ranged from 57 to 268 ng/ml. PR8 was
administered by intranasal instillation in SAL on Day 3, with Ag-THC co-administration
surrounding the day of infection. The rationale for this dosing paradigm was to
investigate the putative affects of Ag-THC treatment on the immune response to PR8

during the early stages of the primary infection.

IV. Effect of A9-THC on viral H1 mRNA levels in wild type and CB."' /CB¢"' mice.
Viral H1 mRNA levels were quantified by real-time PCR, which allows for the
analysis of large numbers of samples in a rapid manner while retaining the specificity and
sensitivity of conventional methods such as the hemagglutination assays (56, 117). A9-
THC treated mice exhibited a higher pulmonary viral H1 mRNA content, that was dose-

dependent and without an effect on mortality, when compared to PR8 infected mice

111

treated with corn oil. These results suggest that Ag-THC administration impairs immune
effectors involved in the clearance of PR8. By 10 dpi H1 mRNA levels approached the
level of detection in all groups, hence suggesting that the clearance of the virus had
occurred in all PR8 treatment groups. In comparison, influenza viral titres have been
shown to return to baseline within 8 days of an uncomplicated influenza infection in
humans (118).

In the comparison of wild type and CBi’/'/CB2'/' mice, we observed greater H]
mRNA levels in the lungs of Ag-THC treated mice challenged with PR8 when compared
to mice challenged with PR8 alone in both wild type and CBi'/’/CB2'/' mice (113). Similar
to our previous observation, we observed H1 mRNA levels in the lungs of A9-THC
treated CB1"'/CB2'" and wild type mice challenged with PR8 that were greater than H1
mRNA levels in CB1'/'/CB2'/' and wild type mice challenged with PR8 alone.
Interestingly, H1 mRNA levels were reduced in the lungs of CBi"'/CB2'/' mice by two
orders of magnitude when compared to H1 mRNA levels observed for wild type mice.
The profound reduction in H1 mRNA levels suggests that the cellular environment that
supports viral entry or growth in the airway epithelium has been impaired in CB1'/'/CBz'/'
mice when compared to wild type mice, or that the kinetics of the cell-mediated immune
response are markedly enhanced in CBi'/'/CB2'/' mice. Our findings of increased
background levels of CD4+ T cells in CB1‘/'/CB2'/' mice, in the current study, support the
latter explanation by suggesting that a hyper-responsive immunity toward PR8 infection
brought about by the absence of functional cannabinoid receptors CB] and CB2 exists.
Moreover, since cannabinoid-mediated effects occurring through CB] and CB2 receptors

on the immune system are most often associated with the suppression of immune

112

responses, the results presented herein would be consistent with the idea that CB1 and
CB2 receptors play a role in dampening immune responses to maintain immune

homeostasis.

V. Effect of PR8 on inflammatory cell recruitment to the pulmonary airways in the
presence or absence of A9-THC in wild type and CBi'" /CB2"' mice.

Inflammatory cells entering the airways of PR8 infected mice followed a classic
pattern of inflammation, in which an early pro-inflammatory response, represented by
neutrophilic influx followed by monocytes and macrophages, was observed early at 3 and
7 dpi, respectively, and a delayed host-immune response, characterized predominantly by
an influx of lymphocytes and to a lesser extent eosinophils peaked by 10 dpi.

A9-THC treatment did not influence the total number of leukocytes retrieved in
BALF between mice challenged with PR8 in the absence and presence of A9-THC
treatment. In spite of trends toward decreased total leukocytes in BALF with Ag-THC
treatment, further analysis of differential cell counts indicated that Ag-THC treatment
decreased, in a dose-related manner, the number of macrophages and lymphocytes in the
airways. Berdyshev and coworkers (119) have previously reported similar findings with
the dose-dependent modulation of immune cell recruitment to the lungs by Ag-THC
following endotoxin exposure.

In light of the observation that Ag-THC modulated the differential leukocyte
counts present in BALF, we further quantified the absolute number of CD4+ and CD8+
lymphocytes. Interestingly, Ag-THC administration attenuated both CD4+ and CD8+ cell

counts by 2-fold. It is noteworthy that measurements of BALF primarily provided

113

information on the cellular and soluble mediators present in the alveolar air space and not
from cells within the alveolar tissue. Given the marked lymphocytic and monocytic
infiltration of the airway submucosa, it was important to consider these data in
conjunction with histopathology, which suggest that Ag-THC modulated the immune
response to PR8 through an influence on leukocyte migration. These findings are
consistent with the effects of cannabinoids on lymphocyte and macrophage chemotaxis
reported by others ( 120-123).

In the comparison between wild type and CBi‘/'/CB2‘/‘ mice, the magnitude of
total leukocyte recruitment to the pulmonary airways and the cellular subsets present
were similar between CB1'/’/CB2’/' and wild type mice challenged with PR8, in the
absence of Ag—THC treatment, suggesting that mice lacking functional CBi/CBz receptors
were capable of mounting an aggressive immune response to PR8 infection. Differential
cell counts provided evidence that the immune response to PR8 by CBi'/'/CB2'/' mice was
more robust or efficient than the immune response mounted against PR8 in wild type
mice. More specifically, the BALF-associated leukocytic subsets of macrophages and
neutrophils were notably decreased in Ag-THC treated CB1'/'/CB2'/' mice infected with
PR8. The decreased recruitment of macrophages and neutrophils to the airways might
point to a cannabinoid receptor dependent effect on the chemotaxis of these leukocytes.
Contrary to findings with macrophages and neutrophils, there were remarkable
similarities in the total number of BALF-associated lymphocytes among all treatment
groups for CBi'/'/CBz'/' and wild type mice. Upon further examination of the T cell
subsets retrieved in BALF, there was evidence that the T cell response to PR8 challenge

with A9-THC treatment was predominantly CD4+ T cells in CBi'/'/CBz'/' mice. As

114

 

mentioned previously, increased numbers of CD4+ T cells might suggest hyperresponsive
immunity in CB1'/'/CB2'/' mice. However, the balance of the T-helper subset population,

and the level of activity of these cells in the pulmonary airways remain unresolved.

VI. Effect of PR8 on the release of soluble mediators into the pulmonary airways in
the presence or absence of A9-THC in wild type and CBi'" ICBz'l‘ mice.

In addition to replicating in the epithelial cells of the respiratory tract, influenza
also infects monocytes/macrophages and other leukocytes by binding to sialic acid
receptor moieties on these cell types (28). Virus infection activates several transcription
factors within these cells that are involved in the induction of chemokine and cytokine
gene expression. These include nuclear factor kappa B (NF -ch), interferon regulatory
factors (IRFS), activating protein (AP)-1, signal transducers and activators of
transcription (STATS) and nuclear factor-interleukin 6 (NF-IL-6 or C/EBPB) (36).
Therefore, influenza plays a direct role in influencing the chemokine and cytokine
makeup of the inflammatory response (reviewed by Julkunen (2001) (36) and Julkunen
(2000) (29)). In these studies, the BALF-associated concentrations of MCP-l, TNF-a,
IL-6, and IFN-y were primarily affected by PR8 infection of mice as compared to mice
instilled with SAL. MCP-l is a chemokine that can be derived from either influenza-
infected epithelia or infected monocytes/macrophages. lL-6 and TNF-a are pro-
inflammatory cytokines produced predominantly by monocytes/macrophages. Instead of
participating in an anti-viral capacity, TNF-a has been suggested to be a driving force for
MCP-l expression. IL-6 serves as a differentiation factor for lymphocytes and stimulates

immunoglobulin production by B cells. Interferon-y, produced by NK cells and/or TH]

115

 

cells, enhances the overall development of cell-mediated immunity, macrophage
activation, antigen presentation, and chemokine gene expression. In these studies, each of
these cytokines was Significantly elevated at 7 dpi.

As Shown in the kinetic study (115), the immune response to PR8 treatment
resulted in increased levels of lL-6, TNF-a, IFN-y, MCP-l and IL-10 by 7 dpi in BALF
in the study that examined the dose-related effects of Ag-THC. Collectively, no clear or
obvious profile of activity emerged concerning the effects of Ag-THC on PR8 cytokine or
chemokine induction. This should not be altogether surprising as each cytokine and
chemokine is regulated in its own unique manner with its own distinct kinetics. In
addition, the molecular mechanism by which certain cytokines and chemokines are
modulated by cannabinoids is only partially understood. In spite of this, several of the
soluble inflammatory mediators evaluated in this study did exhibit dose-dependent
modulation in response to Ag-THC treatment. Specifically, treatment of PR8 infected
mice with Ag-THC at 50 mg/kg and 75 mg/kg led to increased levels of MCP-l and IL-6
in the BALF, whereas IL-10 concentrations were decreased at these dosage levels of A9-
THC. The modulation of these cytokines in response to cannabinoids is supported by
previous findings in other experimental models (121, 124).

In the comparison of wild type and CB1'/'/CB2'/' mice, we observed increased total
BALF associated protein in wild type mice challenged with PR8 that was mildly
attenuated in Ag-THC treated wild type mice infected with PR8. Interestingly, total
protein levels in BALF from CBi"'/CB2'/' mice challenged with PR8 alone were Similar
to wild type mice challenged with PR8 alone, yet were enhanced in Ag-THC treated CBIJ’

/CB2"' mice challenged with PR8. Comparatively, a marked difference existed between

116

Ag-THC treated CB1'/'/CB2'/' and wild type mice infected with PR8, suggesting an
increased severity in either vascular leakiness or inflammatory cell secretions in CBi'I'
/CB2'/' mice. Consistent with increased amounts of total protein in BALF, inflammation
scores accurately reflected changes observed in vascular leakiness. In addition to total
protein, we also measure the BALF-associated chemokine/cytokine levels to provide
information regarding the activity of leukocytes transient to the pulmonary airways.
BALF-associated concentrations of MCP-l, TNF-a, and IFN-y in CBi'/'/CB2'/' mice
infected with PR8, in the absence of A9-THC treatment were modestly elevated with
respect to PR8 infected wild type mice. There was a trend toward decreased
concentrations of these same cytokines in Ag-THC-treated CB1'/'/CB2'/' mice infected
with PR8; however, the collective findings did not provide evidence for CB1 and/or CB2
receptor-dependent or -independent regulation of the secretion of these cytokines. Since
CB1‘/'/CB2'/‘ mice had a lymphocytic response consisting of greater numbers of CD4+ T
cells, and Ag-THC treatment of CBi'/'/CB2"' mice challenged with PR8 resulted in
decreased concentrations of IFN-y, we also measured the BALF-associated
concentrations of the TH2 cytokines IL-2, 1L-4, and IL-5. It has been suggested that A9-
THC treatment modulates cytokine production in a way that results in decreased THl cell-
mediated immunity and increased TH2 humoral immunity (125, 126). Hence, we explored
the potential for decreased THl- type with concomitantly increased TH2-type cytokines in
BALF. Only the cytokine IL-5 was markedly influenced by PR8 infection in wild type
mice in the presence or absence of Ag-THC. IL-S concentrations were reduced in PR8-
infected CB1'/'/CBz'/' mice in the presence or absence of Ag-THC. Ag-THC treatment of

CBl'/'/CB2”' or wild type mice infected with PR8 exhibited no effect on the levels of 1L5

117

when compared to CBt'/'/CB2’/' or wild type mice infected with PR8 alone. The data
suggest that the increased numbers of CD4+ T cells observed in CB1'/'/CBz'/' mice were
not actively secreting more TH2-type cytokines than wild type mice and that there was
not an imbalance between THl- type and TH2-type cytokines secreted due to A9-THC

treatment in response to PR8.

We further evaluated the magnitude of the inflammatory response to PR8 by
measuring serum chemokine/cytokine levels. The serum profile of the pro-inflammatory
chemokine MCP-l is markedly reduced for CBi'/'/CB2'/' mice challenged with PR8 in the
presence or absence of Ag-THC as compared to wild type mice. The reduction in serum
MCP-l is in stark contrast to elevated MCP-l concentrations for the same treatment
groups observed locally in BALF in CBi'/’/CB2'/' mice. The cytokines TNF-a, IL-6 and
IF N-y were elevated with PR8 challenge in the presence or absence of A9-THC. However,
these cytokines were elevated modestly with respect to the detection limits of the assay.
To glean a better understanding of the possible kinetic contribution of these
chemokines/cytokines to the status of immune effectors responding to PR8, their
combined serum and BALF concentrations were considered. The combined local and
serum concentrations for MCP-l following PR8 challenge in the presence or absence of
A9-THC suggest that macrophage recruitment Signals are localized to the pulmonary
airways in CBi'/‘/CBz'/' mice and are more peripherally abundant in wild type mice.
Furthermore, macrophage activity, as measured by TNF-a secretion, is enhanced both
locally and peripherally with PR8 challenge in the presence and absence of Ag-THC in
CB1'/'/CB2’/' mice. More importantly, IFN-y,a cytokine derived from TH] cells, is

markedly elevated in CBi'/'/CB2'/' mice challenged with PR8 alone and suppressed by A9-

118

THC co-treatment. The combined peripheral and local chemokine/cytokine profiles in
CB1'/'/CBp_'/' mice suggest a more vigorous recruitment of leukocytes to the pulmonary

airways following PR8 challenge.

VII. Effect of a single PR8 instillation on pulmonary histopathology in the presence
or absence of A9-THC in wild type and CB." ' /CBz'/ ' mice.

To clarify the relationship between cellular and biochemical findings in BALF
with the dynamic process of airway inflammation, we examined differences in immune
cell recruitment and associated epithelial cell morphology by histopathology. Challenging
mice with PR8 yielded an observed airway epithelial degeneration and necrosis by 3 dpi
with more extensive epithelial regeneration by 7 and 10 dpi, evidenced by the basophilic
nature of the epithelium observed with H&E staining and the positive staining of cellular
nuclei with antibodies directed against PCNA. Epithelial regeneration peaked at 10 dpi,
and resolved by 15 dpi. Coincident with the regenerative process, was an increase in the
total number of epithelial cells enumerated in the PR8 infected group. While epithelial
regeneration was still active at 10 dpi, we observed a metaplastic change of the
epithelium to include more mucus-producing goblet cells that did not resolve by 21 dpi.

Given the marked inflammatory response evident at 7 dpi and the unique finding
of MCM at 10 dpi, we examined the effects of Ag-THC on leukocyte infiltration and
morphologic changes of the airway epithelium associated with influenza infection.
Inflammation scores for histopathology were assigned to assess the influence of A9-THC
on the host immune cell responses to influenza infection. Ag-THC, at all dose levels,

attenuated the magnitude of inflammation at 10 dpi. Most impressively, the

119

histopathology clearly demonstrates that the inflammatory response in the lungs of mice
challenged with influenza alone extends well beyond the perivascular/peribronchiolar
submucosal compartment and into the alveolar parenchyma. With Ag-THC treatment, the
inflammatory response was still centered around the perivascular/peribronchiolar
submucosal compartment; however, the magnitude and severity of inflammation
extending into the alveolar parenchyma was greatly reduced. Therefore, there was less
tissue injury observed with A9-THC treatment.

In the comparison of wild type and CB i'/'/CB2'/' mice, lung sections taken from
the levels of the fifth (proximal) and eleventh (distal) airway generations to the main
axial airway of the left lung lobe were scored for the magnitude and severity of
inflammation present. Within saline instilled wild type mice there was no inflammation
observed. On the other hand, in CBi"'/CB2'/' mice there were 2 out of 8 mice instilled
with saline, in the presence or absence of Ag-THC treatment, which had modest evidence
of inflammatory foci (Figures 8A and 8C). It remains unclear whether the isolated
incidence of inflammation was the result of a subclinical infection in CB i'/'/CB2'/' mice
that went undetected by serological screens or was a spontaneous occurrence of an
underlying pathology in a mouse for which the absence of the cannabinoid receptors CB1
and CB2 might have rendered them immunologically hyper-responsive. Indeed, the CBi'l'
/CB2'/' mice were subjected to an extensive battery of serological screens prior to their
use and were found to be negative for all pathogens tested. Regardless, inflammation
scores were similar for lung sections from CB1'/'/CB2'/' and wild type mice challenged
with PR8, in the absence of Ag-THC treatment. In fact, the histopathology for both PR8-

infected CB1'/'/CB2'/' and wild type mice demonstrated a similar pattern of inflammatory

120

cells infiltrating the perivascular/peribronchiolar submucosa and exhibiting a diffuse
infiltration of the alveolar airspace (Figures 7B and SB). In A9-THC treated wild type
mice challenged with PR8 there was a marked reduction in the inflammation score that
was consistent with our previous finding (113). Also similar to our previous finding was
the localization of inflammation to the submucosa with occasional inflammatory foci
appearing in the alveolar parenchyma (Figure 8D). In contrast, inflammation scores for
lung sections from Ag-THC-treated CB1'/'/CB2'/' mice challenged with PR8 were
modestly enhanced with respect to scores assessed for lung sections from CB1°/'/CB2'/'
mice challenged with PR8 alone. CBi'/'/CB2'/' mice treated with Ag-THC and infected
with PR8 represented some of the most severely affected mice in the study and exhibited
more vigorous inflammatory responses than CBi'/'/CB2'/‘ mice challenged with PR8
alone. Moreover the inflammatory response in Ag-THC treated CB1'/'/CB2'/' mice infected
with PR8 was more pronounced than the respective A9-THC treated wild type mice
challenged with PR8. Also of interest, was the Similarity between inflammation scores
and concentrations of total BALF-associated protein and CD4+ T cells, suggesting that
immune responses to PR8 in the CB i"'/CB2"' mouse are hypersensitive and include

increased vascular permeability and a disproportionate number of CD4+ T cells.

VIII. Effect of a single PR8 instillation on epithelial cell apoptosis and MCM in the
presence or absence of A9-THC in wild type and CBt'l' lCBz"' mice.

Lastly, we examined epithelial cell changes in airways (e.g., apoptosis and MCM)
that occurred during the immune response to PR8. CAS-3 is a well-known marker for

committed activation of apoptosis (127). Apoptosis can be initiated either directly by the

121

virus in infected host cells (128, 129), or by effector cell functions of cytotoxic T cells or
NK cells. PR8 treatment alone enhanced mRNA levels of CAS-3 and tissue staining as
expected. There was a dose-related decrease in CAS-3 tissue staining, but not mRNA
levels with Ag-THC treatment. In the comparison of wild type and CB1'/'/CB2'/' mice,
PR8 challenge led to increased CAS-3 staining as expected in both genotypes; however,
there were no effects observed with Ag-THC administration.

MCM is an adaptive response of the epithelium brought about by soluble
mediators of inflammation. An early indicator of increased mucin production, and
possibly MCM, is the expression of MUC5AC messenger RNA that encodes for the
goblet cell-derived mucin MUC5AC. Mice challenged with PR8 in the absence of A9-
THC exhibited an increase in both the levels of MUC5AC mRNA and alcian blue
staining of tissue, as shown previously (115). Interestingly, A9-THC treatment at a dose
of 25 mg/kg enhanced the levels of MUC5AC mRNA at 7 dpi, whereas the observed
staining of mucosubstances was decreased with 75 mg/kg A9-THC at 10 dpi. Moreover,
the dose-dependent effects of Ag-THC treatment on MUC5AC mRNA at 7 dpi and
mucosubstance staining at 10 dpi were Similar in profile. It should be re-emphasized, that
mRNA levels are reflective of whole lung homogenates, whereas quantification of
morphologic changes in tissue sections (3 days later) is limited in scope to a single
section of the lung. Therefore, the magnitude of the effect elicited by A9-THC is relative
to the measurements made. It is presently unclear whether Ag-THC treatment can directly
interfere with upregulation of MUC5AC gene expression or whether the effect is
mediated indirectly through the suppression of the inflammatory response, more

specifically soluble mediators derived from macrophages and lymphocytes (CD4+ and

122

CD8+ T cells). In the comparison of wild type and CBi'/'/CB2'/' mice, we surprisingly
found that CBi'/'/CB2'/' mice had significantly greater numbers of goblet cells lining the
airways at G5 when treated with Ag-THC alone at 7 dpi, suggesting that Ag-THC is
potentially interacting directly with a signaling pathway tied with increased mucin

production or mucous cell metaplasia in CB1" '/CB2'/' mice.

VIII. Significance and relevance

The body of work presented in this dissertation is a significant contribution to our
understanding of Ag-THC and host-immunity to influenza infection. The present project
characterized time—dependent epithelial and leukocyte responses to a low titre PR8
infection of the pulmonary airways of mice. A rather unique finding in our
characterization was time-dependent PR8-induced MCM of the bronchiolar epithelium.
Although, MCM has been previously reported for adenovirus (23), respiratory syncytial
virus (RSV) (24) and influenza virus (25) infection, the study conducted as a part of this
project was the first to characterize the adaptive epithelial response using morphometric
analysis of the epithelium and cytometric bead array technology to examine a panel of
cytokines that have been implicated in its etiology.

A second unique contribution of these studies was the use of orally administered
Ag-THC. Typically, A9-THC has been administered either intraperitoneally or
intravenously for host immunity Studies. These routes of administration result in larger
circulating concentrations of Ag-THC, initially. The circulating levels of A9-THC
measured in the present studies were in the ng/ml range for Ag-THC and its metabolites.

The concentrations of Ag-THC measured were a reflection of the poor absorption of A9-

123

THC from the gastrointestinal tract following oral administration. Nevertheless, it was
interesting that these low circulating levels of Ag-THC were still capable of altering host-
immunity, in particular leukocyte recruitment, to result in increased levels of viral H1
mRNA.

Another unique aspect of these studies was the analysis of viral burden by
quantitative real-time RT-PCR of the H1 gene. Traditionally, viral burden is measured by
the Madin-Darby Canine Kidney (MDCK) cell assay. The difficulty with the MDCK
assay is that it is not practical for a large number of samples and is not sensitive enough
to distinguish between small differences in burden brought about by the introduction of
another variable, like A9-THC treatment. Quantitative PCR-based methods have been
Shown to have greater sensitivity and Specificity as compared to conventional methods
for quantifying influenza virus, such as hemagglutination assays (56, 117). Moreover,
RT-PCR allows for the analysis of large numbers of samples in a rapid and sensitive
manner. Accordingly, the detection of viral H1 message in these studies demonstrated
that A9-THC administration influenced viral H1 mRNA levels in a dose-dependent
manner.

Finally, these studies employed the use of CBi'/'/CB2'/' mice to elucidate the role
of the cannabinoid receptors CB1 and CB; in the modulation of immune and epithelial
cell responses to PR8. Prior to the generation of CB1‘/'/CB2'/' mice, the identification of
CB1 and CB2 receptor-mediated events brought about by Ag-THC treatment were
assessed by utilizing cannabinoid receptor antagonists for CB1 and CB2 (e.g., SR141716
and SR144528, respectively). However, these cannabinoid receptor antagonists have also

been Shown to have partial and inverse agonist properties (130-132). Since the

124

generation of CB 1" '/CB2'/ ' mice, studies from our laboratory have begun to address the
CB1 and CB2 receptor-independent actions for the suppression of the expression of
cytokines IL-2 and IFN-y by the endocannabinoid 2-arachidonyl glycerol (83, 133). In
the current studies, wild type and CB1'/'/CB2"' mice infected with PR8 in the presence or
absence of Ag-THC demonstrate similarities in detectable levels of viral H1 mRNA and
leukocyte recruitment, suggesting a CB1/CB2 receptor-independent mechanism of
increased viral H1 mRNA content. However, the magnitude of expression of viral H1
mRNA was reduced by several orders of magnitude in CB1'/'/CB2'/' mice and there were
markedly greater numbers of CD4+ T cells in the receptor-null genotype, suggesting that
the kinetics for immune-mediated viral clearance were altered due to the lack of CB1/CB2
receptors. Therefore, the latter finding emphasized a CBt/CBz receptor-dependence for
normal leukocyte recruitment and activation. In addition, another finding unique to the
CB1'/'/CB2’/' mice was the increased numeric cell density of mucin-positive epithelial
cells with Ag-THC treatment alone. The induction of MCM by A9-THC in the absence of
PR8 challenge and, more importantly, in the absence of CBi/CBz receptors indicates that
there are, indeed, other CB1/CB2 receptor-independent activities commenced by treatment
with Ag-THC.

This body of work is a relevant contribution to our understanding of the potential
human health impacts of exposure to chemical agents that render individuals more
susceptible to infection. In these studies we demonstrated that the use of an oral dose of
Ag-THC, that produced serum levels of the parent compound in mice comparable to those
found in humans, decreased host-immunity (e.g.; decreased leukocyte recruitment) to

influenza virus while leading to increased levels of H1 mRNA in the lungs. This finding

125

is important in light of recent concerns regarding the potential for pandemic influenza
infections and the susceptibility to infection of individuals who lack competent immune
systems.

In the last four years attention has been drawn to the newly emergent H5N1
influenza strain that has killed millions of birds in Asia. This virulent strain has jumped
Species by infecting and killing a small number of people in Asia. Currently, the H5N1
strain has not mutated enough to allow for the efficient human-to-human transmission.
However, it is noteworthy that the influenza pandemic of 1918 similarly started as a bird
flu. Unlike previous pandemics, the threat of spreading influenza virus globally with
greater speed has been enhance by our advances in travel. Therefore early detection has
become a priority amongst health organizations like the Centers for Disease Control and
the World Health Organization. In addition, people regarded as belonging to a group of
individuals that are more “susceptible” to influenza have been encouraged to seek early
vaccination. However the term “susceptible” is nebulous, and based on current
guidelines, includes individuals with chemical-induced immune suppression.

Within the last two decades we have witnessed, or in some states voted on,
legislation dealing with the use of marijuana for medicinal purposes. This issue has been
brought to our attention by advocates of medical marijuana for persons suffering from the
adverse wasting effects of acquired immune deficiency syndrome (AIDS) or from nausea
resulting from AIDS or chemotherapy (for treatment of AIDS or cancer). In 1985, the
Food and Drug Administration approved the drug Marinol, a synthetic Ag-THC, for the
treatment of these symptoms; however, patients have contested that the effects of this

pure psychoactive drug rendered them incapacitated. Although patients suffering from

AIDS or cancer may claim a therapeutic benefit from marijuana or its components, little
is known regarding increased susceptibility of AIDS or cancer patients consuming
Marinol or marijuana to infection by respirable pathogens, in light of an already
compromised immunity. This is an important point Since one of the two known
cannabinoid receptors (CB2) is found on immune cells, and cannabinoids have been
shown in human and animal-derived immune cells to be immune suppressive. Therefore,
even within the general population, recreational users of marijuana may be placing
themselves at an added risk for a respiratory infection.

Given the increased viral burden resulting from treating PR8-infected mice with
Ag-THC, we explored the role of CB1 and CB; receptors in regulating Ag-THC-mediated
immune suppression. The observations in these studies clearly point to the importance
these receptors have in maintaining homeostatic immune responsiveness. These studies
also provide evidence that there are other potential receptor targets for A9-THC. For
example, the observation of increased MCM in CB1'/°/CB2'/' mice treated with Ag-THC
may provide a tool with which to evaluate Signaling pathways linked with this

metaplastic event in bronchiolar epithelium.

127

10.

ll.

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139

 

APPENDIX

1. Experimental design for examining the magnitude and severity of inflammatory
response in mouse left lung lobes.

 

 

 

 

Concentration Number
of PR8 of mice
50 pfu 3
300 pfu 3
500 pfu 3

 

 

 

 

All animals were intranasally instilled with 50 ul of PR8 at concentrations of 50, 300, or
500 pfu. Animals were sacrificed 13 dpi. Lungs were inflation fixed with formalin and
microdissected according to the material and methods. Sections of G5 and G11 were

stained with H&E for analysis.

2. Experimental Design for examining the blood concentrations of Ag-THC and its
metabolites ll-hydroxy-A9-THC and 9-carboxy—A9-THC.

 

 

 

 

 

 

 

Dose Number
of THC of mice
0 mg/kg 4
5 mg/kg 4
75 mg/kg 4

 

All animals were orally gavaged with either corn oil or Ag-THC for 5 consecutive days.
On the fifth day, 4 h after the last dose of Ail-THC, the animals were anesthetized with
4% isoflurane and blood was removed from the animals by cardiac puncture. Dr. Jim

Klaunig’s laboratory at IUPUI analyzed the blood samples for Ag-THC and its

metabolites.

140

 

3. Experimental Design for examining the time-dependent changes in pulmonary

responses to PR8.

 

 

 

 

 

 

 

 

 

 

 

Intranasa] Sacrificed Number

Treatment (dpi) of mice
Saline 3 6
Influenza 3 6
Saline 7 6
Influenza 7 6
Saline 1 O 6
Influenza 1 0 6
Saline 1 5 6
Influenza l 5 6
Saline 2 1 6
Influenza 2 1 6

 

 

 

 

 

All animals were intranasally instilled with 50 ul of saline or PR8 at a concentration of
50 pfu. Animals were sacrificed at 3, 7, 10, 15, and 21 dpi. Lungs were lavaged with 2 ml
of sterile saline and then inflation fixed with formalin and microdissected according to
the material and methods. Sections of G5 and G11 were stained with H&E, AB/PAS, and
PCNA for analysis. Lavage was quantified for total and differential cell counts, protein,

elastase, and cytokines by cytometric bead array.

141

 

4. Experimental design for examining the concentration-dependent effects of A9-
THC on pulmonary responses to PR8 (3 studies).

 

 

 

 

 

 

 

 

 

 

 

 

 

Oral Intranasa] Sacrificed Number

Treatment Treatment (dpi) of mice
Corn oil Saline 7 5
Corn oil Influenza 7 5
THC 75 mg/kg Saline 7 5
THC 25 mg/kg Influenza 7 5
THC 50 mg/kg Influenza 7 5
THC 75 mg/kg Influenza 7 5
Corn oil Saline 10 5
Corn oil Influenza 10 5
THC 75 mg/kg Saline 10 5
THC 25 mg/kg Influenza 10 5
THC 50 mg/kg lnfluenza 10 5
THC 75 mg/kg Influenza 10 5

 

 

 

 

 

 

Three separate experiments with this design were conducted. All animals were orally
gavaged with corn oil or 119-THC (25, 50, or 75 mg/kg) and intranasally instilled with 50
pl of saline or PR8 at a concentration of 50 pfu on the third day of gavage 4h prior to
gavage. Animals were sacrificed at 7 and 10 dpi. In one experiment, lungs were lavaged
with 2 m1 of sterile saline. Lavage was quantified for total and differential cell counts,
protein, elastase, and cytokines by cytometric bead array. In the second experiment, the
lungs were inflation fixed with formalin and microdissected according to the material and
methods. Sections of R1-4, G5 and G11 were stained with H&E, AB/H for analysis. In
the third experiment, the entire lung was immersed in TRI reagent and total RNA was

isolated for H1, MUC5AC, and CAS-3 analysis.

142

5. Experimental design for examining the role of cannabinoid receptors in mediating
the effects of A9-THC on immune cells and mediators following PR8 challenge (1"

study).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ora] Intranasa] C5 7Bl/6 Sacrificed Number

Treatment Treatment genotype (dpi) of mice
Corn oil Saline wild-type 7 8
Corn oil Influenza wild-type 7 8
THC 75 mg/kg Saline wild-type 7 8
THC 75 mg/kg Influenza wild-type 7 8
Corn oil Saline CBl(—/-)/CB2(-/-) 7 8
Corn oil Influenza CBl(-/—)/CB2(-/-) 7 8
THC 75 mg/kg Saline CB1(-/-)/CBZ(-/-) 7 8
THC 75 mg/kg Influenza CBl(-/-)/CBZ(-/-) 7 8

 

 

 

Experimental design for examining the role of cannabinoid receptors in mediating
the effects of A9-THC on immune cells and mediators following PR8 challenge (2"d
study).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ora] Intranasa] C5 781/6 Sacrificed Number

Treatment Treatment genotype (dpi) of mice
Corn oil Saline wild-type 7 5
Corn oil Influenza wild-type 7 10
THC 75 mg/kg Saline wild-type 7 5
THC 75 mg/kg Influenza wild-type 7 10
Corn oil Saline CBl(-/-)/CBZ(-/-) 7 5
Corn oil Influenza CB1(-/-)/CB2(-/-) 7 10
THC 75 mg/kg Saline CB1(-/-)/CBZ(-/-) 7 5
THC 75 mg/kg Influenza CB1(-/-)/CBZ(-/—) 7 14

 

 

Using the first experimental design, all animals were orally gavaged with corn oil or A9-
THC (75 mg/kg) and intranasally instilled with 50 pl of saline or PR8 at a concentration
of 50 pfu on the third day of gavage 4h prior to gavage. Animals were sacrificed at 7 dpi.
In the first experiment, right lung lobes were tied off and immersed in TRI reagent for the

isolation of total RNA and the measurement of H1 mRNA. The left lung lobe was

143

lavaged with 1 ml of sterile saline and then inflation fixed with formalin and
microdissected according to the material and methods. Lavage was quantified for total
and differential cell counts, protein, elastase, and cytokines by cytometric bead array. The
left lung lobe was sectioned at G5 and G11 and stained with H&E, AB/PAS for analysis.
Using the second experimental design, the entire lung was immersed in TRI reagent and

total RNA was isolated for H1 analysis.

144

 

   

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