Toxicology of the LeukonDr R B Cope BVSc BSc(Hon 1) PhD cGLPCP DABT ERT

Learning Objectives

• To understand the key basic functional concepts of the leukon;

• To understand the fundamentals of the kinetics of the leukon;

• To understand and accurately interpret changes in the status of the leukon;

• To understand and accurately interpret changes to leukon morphology;

• To understand and accurately recognize important toxicological effects on the leukon

Components of the Peripheral Blood Leukogram• Consists of:– Granulocytes• Neutrophils• Eosinophils• Basophils

– Mononuclear cells• T lymphocytes• B lymphocytes• Other lymphocytes• Monocytes

Neutrophil

Eosinophil

Basophil

Basophil

Monocyte

Neutrophils

Neutrophils

Neutrophils

Neutrophils

Neutrophils• Bone marrow storage pool– Consists of metamyelocytes, bands and segmented N0

– Cells cannot replicate

– Cells are functionally mature despite the differences in morphology

Neutrophils• Bone marrow storage pool– Normal transit time in the SP is 2 – 3 days but this can

shorten considerably on increased demand

– In health, ~ 80% of N0 are in bone marrow are in the storage compartment – a bout a 5 day supply of N0 under normal conditions

– Release from the SP is ordered – oldest cells (segmented N0) are released first

– During increased peripheral demand for N0, younger cells (bands, and in extreme cases metamyelocytes) are released – this is called a “Left Shift”

Segmented N0 in a relatively normal smear

Neutrophilic leukocytosis with left shiftNote that all the N0 in the field are early stage band N0

Neutrophils• Circulating and Marginal Pools– N0 move more slowly in the post-capillary venules than

RBCs due to the presence of adhesion molecules on the N0 and on the endothelium of the post-capillary venules – this population is referred to as the marginated pool

– In the axial or central blood flow of blood vessels make up the circulating pool

– Net result is an uneven distribution of N0 in the circulation: the circulating pool of unadhered N0 and the marginal pool of N0 that are have adhered or are in the process of adhering to the vascular endothelium in the post-capillary venules

Neutrophils

Neutrophils• Circulating and Marginal Pools– Average transit time of a N0 in the circulation is ~ 10 hours

under normal conditions

– N0 subsequently migrate into the tissues. The migration is unidirectional (i.e. they do not re-enter the circulation)

– Some N0 are lost across mucosal surfaces and in secretions

Neutrophils• Mechanisms of neutrophilic leukocytosis (increase in WBC primarily due to an increase in N0)

– Increased stem cell recruitment• Normal response to inctreased demand for N0• Takes 3 – 5 days to have an effect

– Increased effective granulopoesis• Due to an increased number cell divisions in the

development/maturation pool• Also occurs due to decreased death of meylocytes in

the development/maturation pool• Takes 2 – 3 days to have an effect

Neutrophils• Mechanisms of neutrophilic leukocytosis (increase in WBC primarily due to an increase in N0)

– Shortened marrow transit time

– Shift of mature N0 from the marginated pool in the peripheral blood vessels to the circulating pool of mature N0. This results in a neutrophilia without a left shift. This commonly occurs in combination with a reduced number of lymphocytes (lymphopenia) and a reduced number of eosinophis (eosinopenia) in the peripheral blood. This response is referred to as a “stress leukogram.” May be accompanied with lymphopenia, and eosinopenia.

Stress leukograms are caused by adrenalin or cortisol release.They are a common finding associated with exercise/excitement handling and blood collection in many species.The majority of stress leukograms are NORMAL

Causes of neutropenia arranged according to the compartment with which the pathophysiologically relevant mechanism is linked. One should begin the diagnostic approach to a neutropenic patient by seeking to identify the pathophysiologically relevant compartment. Management of a neutropenic patient whose neutrophil production is reduced is entirely different from that of a neutropenic patient whose production is normal and in whom the rate of delivery to the extravascular compartment is normal or appropriately increased in the context of acute infections.

Idiosyncratic Toxic Neutropenia

• Rare but extremely important – potentially lethal agranulocytosis (profound depletion of N0)

• Occurs sporadically within a population• MOA is not the same as the pharmacologic properties of the

xenobiotic• Preclinical tox studies rarely identify or predict this problem• May or may not be dose-related

Idiosyncratic Toxic Neutropenia• MOA– Dose responsive disruption of N0 precursor cell division in

the bone marrow– Non-dose responsive immune-mediated destruction of N0

or N0 precursors – common effect of many drugs, more common in older patients, more common in women, involves anti-N0 antibodies

• Typically involves a sudden decrease in circulating N0 (why?)• Typically persists as long as the xenobiotic is present

• Greatly increases the risk of infection and sepsis if the neutropenia is severe

Idiosyncratic Toxic Neutropenia• Important causes are:– Penicillins– Gold– Clozapine (very aggressive)– Phenothiazines– Purine and purine analogs– Azoles– BZPs

Phagocyte Function

• Neutrophils and monocyte-macrophages are the key phagocytes of the innate immune system

• Their principal innate immune role is to recognize and eliminate microorganisms that make their way past primary physical barriers, such as the epithelium and body secretions that protect the external and lining surfaces of the body.

• Monocyte/macrophages carry out sentinel duty looking for microbes in healthy tissue and act as a bridge between the innate and adaptive immune systems

Phagocyte Function

• Neutrophils appear only in infected or damaged tissue after being recruited by inflammatory mediators released from activated macrophages and endothelial cells or by chemical signals released by invading microorganisms themselves

• After accumulation of these key immune cells at sites of infection, the microbes are eliminated through the process of phagocytosis, which is defined as the engulfment, internalization, and degradation of extracellular material.

Phagocyte Function• Processes involved with N0:– Receptor-mediated adherence to the blood vessel

endothelial wall (shift from the circulating pool to the marginated pool of N0)

– Diapedesis – movement through the blood vessel wall

– Chemotaxis – movement up a chemical concentration of a chemoattractant (endogenously produced and produced by bacteria)

– Binding of the chemoattractant to the N0 cell membrane activation phagocytosis degranulation/oxidative burst/killing

Eosinophils• Important in Type I (IgE-Mast Cell-histamine-mediated)

allergic reactions ( e.g. asthma)• Important in the innate immune response to helminth

parasites • Phagocytic and bacteriocidal capacity resemble that of N0• Both circulating and marginated pools exist• Peripheral blood eosinophilia is generally associated with:– Helminth infections– Allergic processes– Neoplasia (classically mast cell tumors)– Eosinophilic leukemias– Some drugs (e.g. penicillin)– Idiopathic

Eosinophilia-Myalgia Syndrome

• Associated with:– Eosinophilia– Intense myalgia (muscular pain)– Eosinophilic fasciitis– Capillary cell hyperplasia

Eosinophilia-Myalgia Syndrome• Incurable and sometimes fatal syndrome associated with

ingestion of L-tryptophan or substances metabolized to L-tryptophan

• Resembles Spanish toxic oil syndrome due to contaminated rapeseed oil

• Potentially due to a contaminant - 1,1-ethylidenebis (L-tryptophan) (tryptophan dimer)

• Potentially due to a metabolite – 4-aminophenol• Associated with:

– Eosinophilia– Intense myalgia (muscular pain)

Basophils and Mast Cells• Basophils and mast cells have different lineages but similar

functions• Important in Type I hypersensitivity and allergy• Effects are rate• Basopenias

– Acute hypersensitivity reactions (early stages)– Glucocorticoids/stress– Hyperthyroidism

• Basophilias– Chronic allergy/hypersensitivity reactions– Diabetes mellitus– Estrogen– Hypothyroidism– Iron deficiency– Neoplasia

Monocytes

Monocytes

• Derived from bone marrow monoblast.

• Circulate in blood for 1 – 3 days and then move into the tissues

• ~3-8% of blood leukoctes

• Significant storage pool in spleen (~50% of total body monocytes) in the Cords of Biltrothare produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body.

• Following migration from blood, monocytes mature into either tissue macrophages or dendritic cells.

Monocytes• Form part of the immune system – 3 main functions:

– Antigen presentation– Phagocytosis– Cytokine production

• 3 classical types of types of monocytes in blood:

– the classical monocyte (CD14++ CD16- monocyte)– Non classical monocytes (CD14+CD16++ monocyte)– Intermediate cells (CD14++CD16+)

– Different classes represent different developmental stages: Classical Intermediate non-classical

– After stimulation with microbial products the CD14+CD16++

monocytes produce high amounts of pro-inflammatory cytokines

Monocytes• Form part of the immune system – 3 main functions:

– Antigen presentation– Phagocytosis– Cytokine production

• Moncytosis – increase in circulating blood monocyte numbers– Infection

– Recovery phase of neutropenia following infection

– Hyperadreocorticism

– Autoimmune reactions

– Neoplasia/leukemia

– Sarcoidosis

– Lipid storage diseases

Lymphocytes

Lymphocytes• Circulating part of the adaptive immune system

• Lymphocytosis is a feature of infection or neoplasia (leukemias)

• Causes of absolute lymphocytosis include:

– Acute viral infections– Acute & chronic bacterial infections– Some protozoal infections– Leukemias

Lymphocytes• Leukemias/lymphomas in mice

– Extremely common spontaneous finding

– Virtually all mouse strains contain endogeous MuLV retrovirus (Type C) sequences

– Most laboratory mice do not have exogenous MuLV because this is controlled because of SPF

Lymphocytes• Lymphopenia

– Stress leukogram/prolonged exercise

– Recent infection

– Cytotoxic/radiomimetic agents

– CD4+ lymphopenia – classically HIV

– Myelodysplasia