BACTERICIDAL FUNCTION OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES

(1)

Supported by U. S. Public Health Service Grants A! 08821 and Al 06931 and the Department of

Pediatrics Training Grant HD 00053-13. Supported in part by The Minnesota Heart Association, the

John and Mary R. Markie Foundation, and the Icelandic Science Foundation. Parts of these studies were

conducted under the sponsorship of the Commission on Streptococcal and Staphylococcal Diseases of

the Armed Forces Epidemiological Board with support by the Medical Research and Development

Command, U.S. Army, under contract DADA-17-70-C-0082.

Delivered at the annual meeting of the American Academy of Pediatrics, Chicago, October 19, 1971.

ADDRESS: Box 296, Mayo Memorial Building, University of Minnesota Health Sciences Center, \lin-neapolis, Minnesota 55455.

PEDIATRICS, Vol. 50, No. 2, August 1972

264

BACTERICIDAL

FUNCTION

OF

HUMAN

POLYMORPHONUCLEAR

LEUKOCYTES

E. Mead

Johnson

Award

Address

Paul G. Quie, M.D.

Department of Pediatrics, University of Minnesota Medical School, Minneapolis, Minneota

ABSTRACT. Serum from most normal persons

contains specific antibodies which react with

corn-mon bacterial species preparing their surfaces so that phagocytosis by leukocytes can take place. The Fab part of these antibodies reacts with im-munologic specificity with antigens on the surface of bacteria. Another part of the imrnunoglobulin molecule termed the Fc portion is activated during the attachment of the Fab portion to bacteria and becomes a site for attachment of bacteria to

recep-tors on the surface of phagocytic cells. This

activ-ity is greatly amplified by heat-labile serum factors. Normally bacteria are rapidly killed by human

polymorphonuclear leukocytes after engulfment

oc-curs. However staphylococci and gram-negative

species of bacteria survive in the leukocytes of

pa-tients with the syndrome “Chronic Cranulomatous Disease of Childhood.”

These patients have suffered recurrent

se-vere infections with bacterial species that are

part of the body’s resident bacterial flora. By

con-trast these patients are not at increased risk to

infection from such pyogenic bacterial species as group A streptococci or pneumococci. The

leuko-cytes from patients with chronic granulomatous

disease produce little hydrogen peroxide during

phagocytosis. Catalase-producing staphylococci

and gram-negative bacteria are not killed, but

hv-drogen peroxide-producing streptococci and

pneu-mococci are killed. A normal metabolic response to phagocytosis as well as release of lysosonial factors

are essential for the bactericidal activity of human

polymorphonuclear leukocytes. Pediatrics, 50:264,

1972, POLYMORPHONUCLEAR LEUKOCYTES,

STAPH-YLOCOCCI-STREPTOCOCCI, IMMUNOCLOBULIN,

RE-CEPTORS, LETJKOCYTES, CHEDIAK-HIGASHI

SYN-DROME, BACTERIAL ENDOCARDITIS,

MYELOPEROxI-DASE.

The recognition of the E. Mead Johnson award is accepted with gratefulness and humility. I am sure that all of you recognize that the achiecements which led to the win-ning oj this highly coveted award from the Academy of Pediatrics are the combined contributions of predecessors, mentors and

co-workers. I want to express my gratitude

to just a few of these people. The late Nich-olas

J.

Giarman demonstrated to me in the Department of Pharmacology at Yale the

joys of discovery that stem from careful experimentation. Lewis W. Wannamaker at

the University of Minnesota provided my

first opportunity for full-time laboratory

in-vestigation and he has continued to be my

scientific mentor. He s/towed me that disci-pline, determination, and persistence are as important in the laboratory as I knew them

to be on the farm. His example of excel-lence, unwavering faith, aggressive support

and unselfish collaboration have been major factors in making possible the work to be described this morning. Our chief of pediat-rics, John A. Anderson, and Robert A. Good provided the fuel of stimulation and

en-couragement. James G. Hirsch at Rockefel-ler University directed my early interest in phagocytic leukocytes. Principal

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[IC. 1. Model for 7S immiinogluhtilin-C as proposed by Porter. Reprinted from reference 26.

and hard work contributed greatly to the studies I shall describe today were Ralph

C. Williams, Jr., and Ronal4 Mess-ner,

pres-ently at the Unkersity of New Mexico,

Throstur Laxdai at the University of Ice-land, John H. Dossett, University of

Penn-sylcania at Hershey and Edward L. Kaplan,

Richard A. Chilgren, and A. Todd Davis at

the Unicersity of Minnesota. Skilled and

good-natured technical assistance was pro-vided over several years by the following ladies: Josephine Brumbaugh, Linda Schneider, Judy Piepkorn, Margaret Box-meyer, Hattie Gray, and Jean Dettloff.

INTRODUCTION

T

HE nieans by which most of us live in

peaceful harmony with our intimate

bacterial associates has stimulated the

cnn-osity of physicians ever since microscopes

revealed the remarkable microbial world

surrounding us. A highly complex

interac-tion of host-factors and bacterial-factors

al-lows a relationship which is usually

advan-tageous to both species. However, when tile

surface of tile host is breeched by bacterial

invasion an immune response is generated

by the host to localize the invaders. It is the

prompt response of polymorphonuclear

leukocytes with potent bactericidal

capac-ity that confines most bacterial invasions to “border skirmishes.”

Bacteria have highly developed defense

Fob

SS

ss

sS

mechanisms which give them initial

advan-tage in host interactions. These include

ex-tracellular toxins (which will not be

dis-cussed in this presentation

)

and surface

factors which make the bacteria repulsive

to approaching phagocytizing leukocytes.

The neutralization of these surface factors

rendering bacteria more acceptable to

phagocytes is termed opsonization. There

are two distinct classes of opsonins in

se-rum: those that are heat-labile which Dr.

Fred Rosen will describe this morning; and

those that are heat-stable. Heat-stable

opso-nins are primarily specific antibody

di-rected against bacterial surface

compo-nents. When heated serum from patients

with bacterial endocarditis is used as opso-nm in in vitro phagocytosis experiments, high dilutions of these sera are actively op-sonic against the infecting strain of bacte-na.’ These heat-stable opsonic antibodies

developed by patients with bacterial

endo-carditis are directed specifically against the surface antigens of the bacteria infecting them. This is in sharp contrast to sera from

normal persons where heat-labile serum

factors appeared to play the major opsonic

role.1 The scra from patients with subacute bacterial endocarditis, therefore, are

excel-lent sources of heat-stable bacterial

opso-nm. It was found that the 75

(

IgG)

im-munoglobulin fractions from these sera

contained high titers of opsonic activity but

Fc

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FUNCTION OF LEUKOCYTES

no opsonic activity could be demonstrated in the separated

(

1gM

)

195 immunoglobulin fraction.’ This was difficult to explain since Robbins et al.’ had previously reported that

1gM antibodies were 500 times more

effi-cient than IgG antibodies as opsonin

against 2 In that study, however,

an in vivo phagocytosis system was used

and complement and other heat-labile

fac-tors were present. When specifically

ab-sorbed fresh serum

(

containing heat-labile factors

)

was added to 195 serum fractions in our in vitro phagocytosis systems, we

were also able to demonstrate the opsonic

potential of 1gM antibodies.3 Heat-labile

factors therefore appear to be a

require-ment for demonstration of the opsonic

ac-tivity of 1gM antibodies. On the other hand,

IgG antibodies, when present in sufficient

concentration in sera from patients with

bacterial endocarditis, are capable of

op-sonic activity in the absence of heat-labile

factors; however, the opsonic potential of

IgG is greatly amplified by heat-labile

fac-tors.3 These studies suggested to us that

op-sonization of bacteria involves more than

“buttering up” the bacterial surfaces or

neutralizing the bacterial antiphagocytic

factors. In addition, an active site for

at-tachment to the phagocytic cell must be

provided during opsonization of bacteria.

Rheumatoid factors are antibodies to

pa-tients’ own altered immunoglobulins which

develop in patients with rheumatoid

arthni-tis and in approximately 50% of patients

with bacterial endocarditis.4 Rheumatoid

factors attach to the Fe part of

immuno-globulin molecules

(

see Figure 1 for model

of IgG ). To determine if this part of the

molecule is involved in attachment of

op-sonized bacteria to phagocytic cells, serum fractions containing rheumatoid factors were tested in the phagocytosis system.

Rheuma-toid factors inhibited the opsonic action of

Ig antibody bound to bacteria suggesting

that the Fe is an active attachment site.5

There was little inhibition of phagocytosis

by rheumatoid factor when heat-labile

Se-rum factors were present, indicating that

complement components or other

heat-la-bile factors also provide attachment sites.5

Since complement-fixation also occurs on

the Fe portion there may be competition

between rheumatoid factors and

conlple-ment components for a particular site on

the Fe part of the antibody molecule.

Fur-ther evidence of a close association between

complement-fixation and activation of an

attachment site on opsonic antibodies was

provided by experiments which showed

that pepsin digestion of antibacterial

anti-bodies destroyed both opsonic and

comple-ment-fixing activities.6 Furthermore, IgA

immunoglobulins which do not fix

comple-ment have no opsonic activity.6 The results of several studies are summarized in Figure

2. All of these lines of evidence support the

concept that when antibacterial antibodies

opsonize bacteria the Fe portion of the

mol-ecule becomes activated for attachment to

receptor sites on polymorphonuclear

leuko-cytes.

Susceptibility to bacterial illness is the

primary threat to life in patients without

heat-stable opsonins

(

patients with

hypo-gammaglobulinemia

)

and in patients with

deficient numbers of mature

polymorpho-nuclear leukocytes. However, it was not

un-til 1966 that a clinical situation w’as

recog-nized where severe illness from common

species of bacteria resulted from

made-quate bactericidal function w’ithin

phago-cytes. Polymorphonuclear leukocytes from

children with chronic granulomatous

dis-ease of childhood were found to

phagocy-tize opsonized bacteria normally but

staphylococci and gram-negative bacteria

survived inside the intact, morphologically

normal leukocytes.7’ ‘ A description of the

clinical manifestations of this disease is pre-sented with the realization that similar

din-ical courses may be observed in disorders of

phagocytic function from a variety of

causes. Lymphadenopathy and

lympha-denitis are the most frequent presenting

signs in this syndrome suggesting that

de-fective polymorphonuclear leukocyte

func-tion results in dissemination of bacteria and

localization in the reticuloendothelial

(4)

Pepso

a.’cccccccc

,:S.

/

Periodote

00,0-a’

55

FIG. 2. Attachments to and alterations of IgC which modify the Fe portion

of the molecule and inhibit opsonization. In the upper right corner the

structure of IgA is depicted, which also has no opsnic activity. Reprinted

from reference 26.

O6

\ ‘ ‘cf’t

), Pt

Rheurnoto,d Facto, Coomb’s Seto

1 cccccccc,

_____

[ I

j

Mercoptoetttonol

___________________

S.’

5.5

_

267

and granuloma formation occur in the skin,

liver, lungs, and gastrointestinal tract of

these patients.#{176}’1#{176}Pulmonary involvement is present in nearly all children with chronic

granulomatous disease. There is hilar

ade-nopathy, bronchopneumonia, and empyema

as well as lung abscess. The rapid clearing of symptoms usually associated with antibi-otic treatment of bacterial pneumonia is

sel-dom observed, and lung infiltrates persist

for weeks in spite of appropriate antibiotic therapy. In certain patients areas of

bron-chopneurnonia resolve into discrete areas

of consolidation termed “encapsulating

pneumonia” which are distinctive and

sug-gests the diagnosis of chronic granuloma-tous disease when seen on x-ray films.”

Osteomyelitis occurs with great

fre-quency in patients with chronic

granuloma-tons disease. Small bones of the hands and

feet and ribs are typically involved,

some-times with fusiform enlargement. Although

considerable destruction occurs there is

minimal sclerosis, presumably because the

cellular response is that of granuloma

for-snation. The bacteria recovered from bone

lesions are usually gram-negative species

such as serratia or aerobacter. Osteomyelitis

may develop in different bones while a

pa-tient with chronic granulomatous disease is

receiving antibiotic therapy but eventually

after many weeks of therapy complete

heal-ing of affected bones occurs.’#{176} The bacterial species recovered from the lesions of these

children are always catalase producers such

as S. aureus, S. epidermidLi, serratia,

aero-bacter and C. albicans or aspergillus.

In-fections with hydrogen peroxide-producing

species such as streptococci and

pneumo-cocci do not produce unusual illness in

E

z

Time (mm)

FIG. 3. Rate of killing S. aureus (strain 502A ) and E. coli by myeloperoxidase deficient leukocytes and normal leukocytes. ( Permission has been granted by the New England Journal of Medicine to

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268 FUNCTION OF LEUKOCYTES

these patients and are normally killed by

their leukocytes.’3 Fungal disease is quite

common as a terminal event which is not

surprising since C. albicans survive in

the chronic granulomatous disease

leuko-cytes.’4

A precise biochemical abnormality

ex-plaining the bactericidal defect in chronic

granulomatous leukocytes has not been

found. Our initial efforts were directed

to-ward quantitation of known bactericidal

factors in leukocytes. Lysozyme levels and

phagocytin activity were found to be

simi-lar in these leukocytes and leukocytes from

normal persons.8 Similar levels of other

ly-sosome-associated enzymes are also present

in normal levels and measurement of the

rate and magnitude of release of

granule-associated enzymes during phagocytosis has

shown no great difference between chronic

granulomatous disease leukocytes and

con-trol leukocytes.15 The engulfment process in

normal human polymorphonuclear

leuko-cytes is associated with anaerobic glycolysis

and ATP consumption. Intracellular events

which result from the engulfment process

are associated with a burst of respiratory

oxidative activity with increased oxygen

consumption and hexose monophosphate

shunt activity. More than 10% of

glucose-i-C is metabolized by the hexose

monophos-phate shunt after engulfment of bacteria.

There is accumulation of hydrogen

perox-ide in phagocytic vacuoles as a result of

these metabolic shifts. Leukocytes from

pa-tients with chronic granulomatous disease

do not respond to phagocytosis with

in-creased oxygen uptake. There is little shift

to hexose monophosphate shunt metabolism

and little accumulation of hydrogen

perox-ide.1#{176}The demonstration by Klebanoff17

that hydrogen peroxide in association with

myeloperoxide

(

a lysosomal enzyme

)

and

halides provides phagocytic cells with

po-tent bactericidal activity suggested that

failure of leukocytes from patients with

chronic granulomatous disease to produce

hydrogen peroxide may be a critical factor

in the failure of these cells to kill bacteria.

This possibility was strengthened by our

discovery that Streptococcus pyogenes,

Streptococcns viridans, and Streptococcus faecalis were rapidly killed 1w leukocytes from patients with chronic granulomatous disease.1 All of these species are hydrogen

peroxide producers. The bacteria

them-selves provide hydrogen peroxide in the

phagocytic vacuoles so that the

rnyeloper-oxidase-hydrogen peroxide-halogen

bac-tericidal system operates efficiently. The

rate limiting factor for hexose monophos-phate shunt activity is NADP + which serves as a hydrogen acceptor for two

dehydroge-nases in the shunt. Although Baehner and

Karnovsky’8 reported diminished NADH

oxidase activity in chronic granulomatous

disease leukocytes, Holmes, et al.’ found

normal levels in their patients. It is hoped

that a cooperative study of the same

pa-tients using the same technique will resolve this controversy.

Leukocytes from the fathers of male

pa-tients have shown normal bactericidal

func-tion but the polymorphonuclear leukocytes

from mothers and some sisters showed

bac-tericidal capacity intermediate between the

patients and controls. When

polvmorpho-nuclear leukocytes from mothers of boys

with chronic granulomatous disease were

incubated with nitroblue tetrazolium

dun-ing phagocytosis approximately 50% of the

polymorphs reduced nitroblue tetrazolium

to a blue formazan precipitate in the

cyto-plasm in contrast with less than 5% in

pa-tients with the disease and 70% to 90% in

normals.’9 This histochemical identification

of two metabolically different cell

popula-tions suggests random inactivation of the X

chromosome in female cells and X linked

genetic transmission. The carriers are not

unusually susceptible to bacterial disease.

Female patients with the syndrome of

chronic granulomatous disease, including

severe, recurrent abscesses and leukocytes

with diminished bactericidal capacity, have

been reported.2#{176} No evidence of a carrier

state in family members of female patients

has been demonstrated so that genetic

transmission via the X chromosome is not

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granu-269

lomatous disease. Defective bactericidal

function of polymorphonuclear leukocytes

may be the basis of recurrent severe

bacte-nial disease with granulomatous lesions in

females as well as males.

There is accumulating evidence that

de-fective leukocyte function may be found in

other clinical conditions. Lehrer and Clin&’ described a patient with congenital absence

of myeioperoxidase and prolonged candida

infection. They demonstrated delayed

bac-tenicidal capacity for E. coli and S. aureus

in the patient’s leukocytes. In addition we

recently studied a patient who exhibited

markedly deficient polymorphonuclear

leukocyte myeloperoxidase associated with

myelomonocytic leukemia and delayed

bactericidal function of his

polymorphonu-clear leukocytes.22 As shown in Figure 3,

there was little bactericidal activity in the

patient’s cells after 30 minutes and 60

mm-utes incubation

(

the control cells had killed

99% of inoculated bacteria at this time)

but after 4 hours’ incubation the patient’s

leukocytes had killed more than 90% of the

bacteria. Alternative bactericidal

mecha-nisms appear to eventually kill intracellular

bacteria in leukocytes deficient in

myelo-peroxidase. Root” has also shown that

pa-tients with Chediak-Higashi syndrome have

an approximate 30-minute lag in initiation of intracellular killing of staphylococci and

gram-negative bacteria. There are

abnor-mal lysosomes in the Chediak-Higashi

syn-drome and myeloperoxidase does not enter

phagocytic vacuoles in normal

concentra-tions even though hydrogen peroxide

accu-mulation is normal.

McCall and co-workers recently reported

that leukocvte bactericidal capacity was

di-minished in patients with severe infections

when peripheral blood smears contained a

high percentage of leukocytes with toxic

granules and D#{246}hle b24 We recently

confirmed this observation in two patients

with severe acute 25 One patient

had multiple staphIococcal abscesses and

the other a severe post-operative wound

in-fection. The leukocytes from these patients

phagocytized S. aureus normally, but there

was prolonged survival of intracellular bac-teria. Clinical progress was slow and defec-tive bactericidal capacity could be

repeat-edly demonstrated over a 5-week period in

one of the patients. The patient’s clinical

improvement coincided with return of

len-kocyte function to normal.

CONCLUSION

Each of the groups of patients discussed

this morning taught us something new

about host resistance to bacterial illness. Patients with bacterial endocarditis showed

us that opsonic antibodies not only

neutral-ize bacterial antiphagocytic factors but

pro-vide an active site for attachment to

phago-cytes. Children with chronic granulomatous disease taught us that the leukocyte’s meta-bolic response to phagocytosis is essential

for bactericidal activity. Hydrogen

perox-ide, a product of this metabolism, is an

im-portant co-factor for halogenation and

kill-ing of bacteria. Patients with diminished

leukocyte myeloperoxidase demonstrated

that this lysosomal enzyme is also a neces-sary co-factor for killing intracellular bacte-na.

Finally, there are many patients with

in-creased susceptibility to bacterial illness in

whom all available studies are normal.

These patients emphasize that we have

much more to learn about our relationships

with our bacterial world.

REFERENCES

1. Laxdal, T., Messner, R. P., Williams, R. C., Jr..

and Quie, P. C. : Opsonic, agglutinating and

complement-fixing antibodies in patients with

subacute bacterial endocarditis. J. Lab. Clin.,

71:638, 1968.

2. Robbins, J. B., Kenny, K., and Suter, E. : The

isolation and biological activities of rabbit

gamma-M and gamma-G anti Salmonella

typhimurium antibodies. J. Exp. Med., 122: 385, 1965.

3. Dossett, J. H., Williams, R. C., Jr., and Quie, P. G. : Studies on interaction of bacteria,

serum factors and polvmorpbonuclear

leuko-cvtes in mothers and newborns. PEDIATRICS,

44:49, 1969.

4. \\‘illiams, H. C., Jr., and Kunkel, H. C. :

Rheu-matoid factors, complement and conglutinin

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bacte-270 FUNCTION OF LEUKOCYTES

rial endocarditis. J. Clin. Invest., 41:666,

1962.

5. Messner, R. P., Laxdal, T., Quie, P. C., and Williams, R. C., Jr. : Serum opsonin, bacteria

and polymorphonudear leukocyte

interac-tions in subacute bacterial endocarditis. J. Clin. Invest., 47:1109, 1968.

6. Quie, P. C., Messner, R. P., and Williams,

R. C., Jr. : Phagocytosis in subacute bacterial endocarditis. Localization of the primary

op-sonic site to Fe fragment. J. Exp. Med., 128:

553, 1968.

7. Holmes, B., Quie, P. C., Windhorst, D. B., and Good, R. A.: Fatal granulomatous disease of childhood, an inborn abnormality of phago-cytic function. Lancet, 1 : 1225, 1966.

8. Quie, P. C., Holmes, B., White, J. C., and

Good, R. A. :In vitro bactericidal capacity of human polymorphonuclear leukocytes: Di-minished activity in chronic granulomatous

disease of childhood. J. Clin. Invest., 46: 668, 1967.

9. Quie, P. C. : Chronic granulomatous disease of

childhood. In Schulman, I., ed.: Advanc.

Pediat, 16:287, 1969.

10. Quie, P. G., Kaplan, E. L., Laxdal, T., and Dossett, J. H. : Phagocyte abnormalities. In Kagan, B. M., and Stiehm, E. R., ed. :

Im-munologic Incompetence. Chicago: Year

Book Med. PubI., pp. 233-243, 1971.

11. Wolfson, J., Laxdal, S. D., Quie, P. C., and Good, R. A. : Roentgenologic manifestations in children with a genetic defect of poly-morphonuclear leukocyte function.

Radiol-ogy, 91:37, 1968.

12. Wolfson, J. J., Kane, W. J., Laxdal, S. D.,

Good, R. A., and Quie, P. C. : Bone findings in chronic granulomatous disease of child-hood. A genetic abnormality of leukocyte

function. J. Bone Joint Surg., S1A: 1573,

1969.

13. Kaplan, E. L., Laxdal, T., and Quie, P. C.:

Studies of polymorphonuclear leukocytes

from patients with chronic granulomatous disease of childhood: Bactericidal capac-ity for streptococci. PEDIATRICS, 41:591,

1968.

14. Oh, M. H. K., Rodey, C. E., Good, R. A., Chil-gren, R. A., and Quie, P. C. : Defective can-didacidal capacity of polymorphonuclear

leukocytes in chronic granulonatous disease of childhood. J. Pediat., 75:300, 1969. 15. Baehner, R. L., Kamovsky, M. J., and

Karnov-sky, M. L. : Degranulation of leukocytes in

chronic granulomatous disease. J. Clin. In-vest., 48:187, 1969.

16. Holmes, B., Page, A. R., and Good, R. A.:

Studies of the metabolic activity of leuko-cyt es from patients with a genetic

abnormal-ity of phagocyte function. J. Clin. Invest.,

46:1422, 1967.

17. Klebanoff, S. J.: Intraleukocvtic nicrobicidal

defects. Ann. Rev. Med., 22:39, 1971.

18. Baehner, R. L., and Karnovsky, M. L. :

Defi-ciency of reduced nicotinamide-adenine

di-nucleotide oxidase in chronic granulomatous

disease. Science, 162: 1277, 1968.

19. Windhorst, D. B., Page, A. R., Holmes, B.,

Quie, P. C., and Good, R. A.: The pattern of genetic transmission of the leukocyte defect in chronic granulomatous disease of child-hood. J. Clin. Invest., 47: 1026, 1968. 20. Quie, P. G., Kaplan, E. L., Page, A. R.,

Grus-kay, F. L., and Malawista, S. E. : Defective polymorphonuclear-leukocyte function and chronic granulomatous disease in hvo female children. New Eng. J. Med., 278:976, 1968. 21. Lehrer, R. I., and Cline, M. J.: Leukocyte

myeloperoxidase deficiency and dissemi-nated candidiasis. The role of mveloperoxi-dase in resistance to candida infection. J. Clin. Invest., 48: 1478, 1969.

22. Davis, A. T., Brunning, R. D., and Qnie, P. C.:

Polymorphonuclear leukocvte myeloperoxi-dase deficiency in a patient with

myelomo-nocytic leukemia. New Eng. J. Med., 285:

789, 1971.

23. Root, R. K., Rosenthal, A. S., and Balestra,

D.

J.: Abnormal bactericidal metabolic and lysosomal functions of Chediak-Higashi syn-drome leukocvtes. J. Clin. Invest., in press. 24. McCall, C. E., Canes, J., Cooper, R., and

De-Chatelet, L. : Functional characteristics of human toxic neutrophils. J. Infect. Dis., 124: 68, 1971.

25. Messner, R. P., Davis, A. T., and Quie, P. C.: Amer. J. Med., in press.

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1972;50;264

Pediatrics

Paul G. Quie

LEUKOCYTES: E. Mead Johnson Award Address