Leukocyte Adhesion

Leukocyte Adhesion - 1

Medical Cell Biology

60:116October 27, 2010

LEUKOCYTE ADHESION

Morris O. Dailey, M.D., Ph.D.

Department of Pathology and The Program in ImmunologyOffice: 145 MRC e-mail: [email protected]

Reading Assignment:

Textbook: Alberts et al., Molecular Biology of the Cell.pg. 1549-51: Overview of normal lymphocyte recirculationpg. 1169-74: Summary of integrin family of cell adhesion moleculespg. 1531-1532: Overview of neutrophil and macrophage response to infectious agents

See also the supplementary material posted on the class web site for a summary of the major concepts.

KEY CONCEPTS:1. Leukocytes circulating in the bloodstream, including lymphocytes, neutrophils, and monocytes, are

important mediators of the host resistance to all infectious agents.2. Leukocytes interact with each other, with the extracellular matrix, and with vascular endothelial cells

via complementary pairs of adhesion molecules.3. Leukocytes migrate continually throughout the host and traffic to sites of infection (the

inflammatory response) using mechanisms involving these specific adhesion systems.4. Defects in leukocyte adhesion adversely affect cell traffic and inflammatory responses and can have

profoundly deleterious clinical effects on the host.

LECTURE OVERVIEW:

Unlike other nucleated cell types, leukocytes continuously traffic throughout the host and migrate into tissues when called upon to destroy foreign material or assist in repair of tissue damage. Regardless of how effective a lymphocyte, for example, might be in killing virus-infected cells in vitro, if they do not move to the site of infection, they will be not have any activity in the whole person. Thus, the ability of leukocytes to move continuously and in a directed manner is critically important for maintaining normal immune responsiveness and natural resistance to infection. In order for leukocytes to migrate into and within any organ, they must adhere to the inner lining of blood vessels and subsequently to extracellular matrix in the tissue. This lecture will provide an overview of the adhesive mechanisms responsible for these processes and illustrate the clinical problems associated with their deficiency.

For a brief description of the different kinds of leukocytes, you may (optionally) review page 1451 through the top of page 1453 in Alberts. A basic understanding of the major leukocyte types is required to understand the material presented in this lecture.


Slide 1Goals of this Lecture

Learn the general principles of how lymphocytes and other leukocytes traffic throughout the body.

Discuss the molecular mechanisms that regulate leukocyte migration in normal and inflammatory states.

Discuss a patient with recurrent infections and abnormal inflammatory responses.

Apply the insights learned here to understanding thepathophysiology underlying this patient’s illness.

This lecture will present to you an overview of leukocyte traffic and the inflammatory response. You will learn much more about it in other courses later.

Slide 2

Body’s localized response to injury

Grossly (on skin):

Overview of Inflammation

Firm, red, tender, swollen

Histologically:

Infiltration of tissue with any of the classes of leukocytes

Neutrophils (acute inflammation)

Lymphocytes (chronic inflammation)

Inflammation: the body’s response to localized infection or injury. Helps kill microorganisms and repair tissue.

The major types of leukocytes (white blood cells):

T and B lymphocytes: mediate antigen- specific immunity.

Neutrophils: Phagocytose bacteria, dead cells & debris. Part of the innate immune system.

Monocytes: also phagocytic, functionally similar to neutrophils.

Slide 3 Leukocyte Traffic

Antigen-non-specific cells: monocytes, neutrophils

Circulate through bloodstream and enter tissues to form inflammation and phagocytose organisms.

Antigen-specific cells: T and B lymphocytes

Circulate through blood and traffic to inflammatory sites.

Also recirculate continuously among all secondary lymphoid tissues.

Recirculation occurs all of the time - it doesn’t require any stimulus. In contrast, inductive stimuli (e.g., bacteria, cytokines) are needed to redirect the flow of leukocytes to sites of inflammation, as described later. This change in traffic is transient, ceasing after the infection or injury has been repaired.

Note that only lymphocytes, but not other types of leukocytes, undergo recirculation (see slides 9 and 10).


Slide 4B l o o d F l o w

A r t e r i o l e s t o C a p i l l a r i e s t o V e n u l e s

L e u k o c y t e s e x i t b l o o d s t r e a m i n s m a l l v e n u l e s

Diagram of blood flow, showing the basic structure of blood vessels and a leukocyte beginning to exit the blood into the surrounding tissue.

Slide 5 Inflammatory Response: Overview

Blood Vessel

EndothelialCells

Basement membrane

Tissue

Leukocytes exit the bloodstream by passing through the walls of small venules.

Basic vessel structure: lined by continuous layer of endothelial cells attached to subjacent basement membrane.

Slide 6Leukocyte Adhesion and Migration

All leukocyte traffic requires adhesion

Between leukocytes and extracellular matrix

Between leukocytes and the surface of apposing cell

Mediated by cell surface adhesion molecules

The expression of adhesion molecules on both leukocytes and endothelial cells is an absolute requirement for all leukocyte traffic, both lymphocyte recirculation through lymph nodes and leukocyte migration into all types of inflammation.


Slide 7 Patient History - SummaryFull-term infant of closely related parents.

Returned 8 days after birth with infection and poor healing around site of circumcision and cellulitis around umbilicus.

Surgery for stenotic segment of bowel followed by bacteremiaand cellulitis at site of i.v. catheter.

For several months numerous infections and very high WBC even when not infected.

Fluid from infected lesions showed numerous bacteria but never any neutrophils.

Died of recurrent infection at 8 months of age.

Cellulitis: spreading bacterial infection in skin and subcutaneous tissue

Bacteremia: bacterial infection in the blood-stream; can be fatal

High white blood cell count: an indication of infection

Slide 8Key Clinical Points

1. Delayed umbilical cord separation

2. Poor wound healing

3. Recurrent bacterial infections

4. Leukocytosis in the absence of infection

5. Paucity of leukocytes in wound drainage

Note:

The separation of umbilical cord after birth is dependent on local inflammation.

Slide 9Constitutive Leukocyte Traffic

Continuous redistribution among lymphoid organs (such

as the spleen, lymph nodes)

Lymphocyte Recirculation

Recirculation occurs all of the time - it doesn’t require any stimulus. In contrast, inductive stimuli (e.g., bacteria, cytokines) are needed to redirect the flow of leukocytes to sites of inflammation, as described later. This change in traffic is transient, ceasing after the infection or injury has been cleared.

Note that only lymphocytes, but not other types of leukocytes, undergo recirculation.


Slide 10 Lymphocyte Recirculation

Arterioles

Thoracicduct

Thoracicduct

HEV

AfferentLymphatic

LymphNode

EfferentLymphatic

Heart

VenaCavaVenaCava

Skin

DermisArterioles

HEV

AfferentLymphatic

LymphNode

EfferentLymphatic

Heart

Skin

Dermis

Diagram of lymphocyte recirculation through lymph nodes. A much smaller component involves lymphocytes first passing through other organs, such as skin, and then traveling through lymphatics to the local nodes.

Slide 11

Scanning electron micrograph of the lymph node high endothelial venule (HEV), showing numerous lymphocytes adhering to the endothelial surface. These small vessels are highly specialized for very high levels of lymphocyte adhesion.

Slide 12 Lymphocyte - Endothelial Adhesion

L-selectin

L-selectin Ligand

in Lymph Node High Endothelial VenulesThe major adhesion receptor-ligand system that mediates lymphocyte traffic to lymph nodes. L-selectin is also important in initiating the movement of cells into inflamed tissues (described later).


Slide 13 Inflammatory Response: Overview

Blood Vessel

EndothelialCells

Basement membrane

Tissue

Example: acute inflammation induced by bacteria in the skin.

Bacteria stimulate secretion of cytokines by cells such as macrophages in the tissue, which diffuse to the endothelium. Here they induce the expression of specific adhesion molecules that can bind to complementary molecules on the neutrophil membrane.

Slide 14

Families of the major leukocyte and endothelial adhesion molecules.

Slide 15

•Transmembrane heterodimersimportantin cell-cell and cell-ECM interactions

•More than 20 members in the family

•Composed of distinct and chains

•Each family defined by its chain

Integrins

LigandBinding

Membrane

The major integrin important for neutrophil adhesion and traffic to inflammation, Mac-1, is composed of one alpha(M) and one beta(2) chain.


Slide 16 Diagram of the Integrin Family of

Adhesion Molecules

Each integrin is composed of one alpha and one beta chain. Most, but not all, alpha chains bind to only one beta chain. A few, such as alpha(4), can bind to two distinct beta chains, the resulting heterodimers having different adhesive specificities (bind to different ligands).

Slide 17 Integrin Family

Expressed on all leukocytes

Bind to ECM proteins, e.g., fibronectin

Bind to counterreceptors induced on activated endothelial cells at sites of inflammation:

Mac-1 on neutrophils & monocytes to ICAM-1 on EC

ECM = extracellular matrix

ICAM-1: discussed later

Slide 18 Regulation of Integrin Function on Leukocytes

Level of expression up regulated on activated leukocytes

Adhesive function (affinity) upregulated by chemokines

Neutrophils: occurs in minutes

Lymphocytes: several days

Occurs in only a few hundred milliseconds

In lymphocytes, monocytes, and neutrophils

The two major mechanisms of modulating integrin-mediated leukocyte adhesion.

Changes in integrin affinity is the most important mechanism of regulating adhesion.


Slide 19 Chemokines

Small polypeptides secreted by activated leukocytes and some other cell types, such as macrophages

Many different chemokines with different target cells

Bind to surface of ECs where they can interact with receptors on circulating leukocytes

Chemotactic for leukocytes (e.g., IL-8 for neutrophils)

Induce up regulation of integrin affinity

Different chemokines are secreted by a wide variety of cell types and mediate many functions in addition to leukocyte adhesion and chemotaxis.

Slide 20 I n t e g r i n A f f i n i t y M o d u l a t i o n

L o w A f f i n i t y S t a t e

H i g h A f f i n i t y S t a t eI n s i d e - o u tS i g n a l i n g

After signaling from within the cell, the conformation of the binding domain of the integrin changes such that it can bind more effectively (i.e., with higher affinity) to the ligand on the apposing cell.

Slide 21 Immunoglobulin (Ig) Superfamily

ICAM-1

Superinduced by cytokines released locally by lymphocytes and tissue macrophages (e.g., IFN and TNF)

Expressed at very low levels on resting EC

(Intercellular Adhesion Molecule-1)

Binds to Mac-1 expressed on both monocytes andneutrophils

Immunoglobulin: antibody molecules. Many proteins have structures related to Ig, including several cell surface adhesion molecules.

ICAM-1 also binds to an adhesion molecule on lymphocytes (LFA-1) that mediates the same type of adhesion as Mac-1 on neutrophils. (You don’t have to remember LFA-1 for now)


Slide 22 Selectin Family

L-selectin

P-selectin

E-selectin

All selectins bind via N-terminal lectin domains to CHO groups on mucin-like counterreceptors.

A set of three structurally and functionally related adhesion molecules.

We will discuss only leukocyte (L)-selectin.

Slide 23 Selectin Family

Constitutively expressed by monocytes, neutrophils, and most lymphocytes

L-selectin

On lymphocytes it mediates adhesion to HEV, homing to lymph nodes, and recirculation

On monocytes and neutrophils it mediates initial stage of adhesion in inflammatory sites.

Ligands are induced on endothelium by cytokines, such as interferon-gamma

The high endothelial venules (HEV) are the specialized venules mentioned earlier through which lymphocytes pass into lymph nodes.

We will henceforth be concerned primarily with the role of L-selectin on neutrophils and monocytes (i.e., in the inflamatory response, not recirculation).

Slide 24

Negatively charged CHO side chains

Lectin domain

SelectinLigand L-selectin on a leukocyte binding to carbohydrate-rich ligand on an endothelial cell.

As you will soon see, this specific type of interaction is a critically important for adhesion of leukocytes flowing in the bloodstream.


Slide 25 Key Features of Selectin-Mediated Adhesion

Very rapid on-rate, characteristic of lectin-carbohydrate interactions

Very low binding affinity, too weak to stop cells in shear

Results in a rolling interaction with endothelium

Integrin-mediated adhesion: slow on-rate but high affinity

Shear-resistant: mediates leukocyte arrest and firm adhesion

Differences between integrin- and L-selectin-mediated adhesion:

Affinity vs. kinetics of binding

Critically important for sequence of leukocyte adhesion.

Slide 26 Cascade of Events in Leukocyte Adhesion and

Transendothelial Migration

Similar for all leukocytes

Show example next for neutrophils

The following four figures show the several distinct steps that must occur sequentially in order for leukocytes to get out of the blood-stream and migrate into the surrounding tissue.

Slide 27

Endothelium

Endothelial Cell Adhesion Molecules Induced

ICAM-1

Selectin ligand

Cytokine Secretion

Bacterial products stimulate cytokine release from cells (such as macrophages) in the tissue. The cytokines diffuse to the blood vessel where they induce the expression of genes coding for the adhesion molecules shown. After a few hours these adhesion molecules become expressed at high levels on the endothelial cell surface.


Slide 28

ICAM-1

Selectin ligand L-selectin

CirculatingNeutrophilTethers to Endothelium

ViaSelectins

Secretion ofChemokines

Initial adhesive interaction: L-selectin with its endothelial ligand - cells slow down and roll across surface.

Chemokines are secreted by several cell types in the tissue; diffuse to endothelium.

Slide 29

Selectin ligand L-selectin

ChemokineReceptor

Chemokine Triggers Integrin Affinity

ICAM-1

Mac-1

and Firm Adhesion

Chemokines on the surface of the endothelium interact with receptors on the neutrophil. This stimulates a cascade of events that results in the inside-out signaling of leukocyte integrins that induces their high affinity state.

The high affinity binding of Mac-1 to ICAM-1 results in the arrest of the flowing neutrophil.

Slide 30 Leukocyte Adhesion and Transendothelial Migration The leukocyte passes through the venule,

between endothelial cells and through the basement membrane. They then migrate through the tissue toward the source of the chemokine, which helps localize the phagocytic neutrophils close to the invading bacteria (or other noxious agent).

Please note the distinct roles played by cytokines (inducing adhesion molecules) and chemokines (affinity up-regulation and chemotaxis) in the inflammatory response.


Slide 31

Requires two sequential types of adhesion:

Key Concepts

Rapid but loose rolling interaction - selectins

Firm adhesion - integrins

Requires local secretion of cytokines to induce the expression of endothelial adhesion molecules

Requires chemokine deposition on endothelial surface to trigger increased integrin affinity and for chemotaxis

Entry of Leukocytes into Tissues

Remember and understand these!

Slide 32

Immunofluorescence showed that neutrophils did not express Mac-1 (CD11b)

Our Case

Neutrophils isolated from the patient did not adhere to plastic in vitro

Leukocyte Adhesion Deficiency Disease (LAD)

Mutations in integrin chain prevents association with chains

No integrin expression on leukocytes

Major deficit is inability of neutrophils to firmly adhere to activated endothelium or undergo transendothelial migration.

Back to a discussion of our case:

The clinical syndrome in our case results from the lack of expression of Mac-1 on the surface of leukocytes, the most important effect being on neutrophil function.

Without Mac-1 expression, neutrophils (and monocytes) cannot adhere tightly to EC and therefore cannot transmigrate out of the bloodstream.

Slide 33

Consequences of inability to adhere to endothelium:

Our Case

Neutrophils cannot exit bloodstream:extremely high neutrophil count in blood

Neutrophils cannot enter infected tissues:no leukocytes at sites of infectionbacteria grow uncheckedpoor wound healing

Leukocyte Adhesion Deficiency Disease (LAD)Review our clinical case again.


Slide 34 LAD Type II

A specific enzyme required for synthesis of the carbohydrate portion of selectin ligands is deficient

No functional L-selectin ligands are expressed

Clinical syndrome similar to but less severe than LAD I

Decreased rolling and inability to recruit leukocytes from the circulation into infected tissues

A similar disease, although not quite as severe as the LAD Type I just described.

Because of the lack of its endothelial ligand, L-selectin cannot mediate the rapid adhesion necessary to slow cells down in the bloodstream. No adhesion can therefore occur, even though integrins are normal in these patients.

Slide 35 Nomenclature

Many different names for most adhesion molecules

Cluster of Differentiation (CD) System:

Mac-1 CD11b/CD18

ICAM-1 CD54

L-Selectin CD62L

The CD nomenclature is a standardized format for cell surface proteins. You will hear more about this in your Immunology course.

For additional explanation of adhesion and inflammation, and its application to this clinical case, please review the Supplemental Handout that will be posted on the class web site.

For a recent overview of leukocyte adhesion in immunodeficiencies, I recommend the following review article: (this is not assigned reading)

Notarangelo, L.D. and Badolato, R. Leukocyte trafficking in primary immunodeficiencies. Journal of Leukocyte Biology volume 85, March 2009, page 335.

Documents

Leukocyte Adhesion