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FACTORS INVOLVED IN PASSIVE TRANSFER OF

CONTACT HYPERSENSITIVITY

APPROVEDi

mmm essor

tf'i M ,<r • , , • „ „ Minpfr Professor 1

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Director o£" ','Depa^^entn" o# feiology

D««ui t)!f Sr«cluat« keto'ooi ^

FACTORS INVOLVED IN PASSIVE TRANSFER OF

CONTACT HYPERSENSITIVITY

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF ARTS

By

Walter L. Ellis, B. A,

Denton, Texas

August 1966

TABLE OF CONTENTS

Page

LIST OF TABLES iv

LIST OF ILLUSTRATIONS * v

Chapter

I. INTRODUCTION 1

II. MATERIALS AND METHODS 7 Animals Chemicals Preparation of Homologous Serum Conjugate Sensitization of Animals Skin Testing Production and Collection of Peritoneal

Exudate Testing for Passive Systemic Anaphylaxis Testing for Presence of Antibody

III. RESULTS 16

Skin Tests of Donor Animals Skin Tests and Passive Systemic

Anaphylaxis of Recipient Animals : im-passive Hemagglutination Test

IV. DISCUSSION 26

V. SUMMARY... 33

BIBLIOGRAPHY 35

xix

LIST OF TABLES

Table Page

I. Skin Tests Reactions . 9

II. Grades of Passive Systemic Anaphylaxis... 11

III. Skin Test Results of Donor Animals..... 17

IV. Skin Test of Lymph Node Recipients 18

V. Skin Tests and Passive Systemic Anaphylaxis of Recipients Receiving Peritoneal Cells After Incubation in Hanks 19

VI. Skin Tests and Passive Systemic Anaphylaxis of Recipients Receiving Supernatant After Incubation in Hanks of Peritoneal Exudate Cells 21

VII. Skin Tests and Passive Systemic Anaphylaxis of Recipients Receiving Peritoneal Exudate Cells After Incubation with Serum Conjugate.. 22

VIII.B Skin Tests and Passive Systemic Anaphylaxis of Recipients Receiving Supernatant After Incubation of Peritoneal Exudate Cells with Serum Conjugate... 2 3

IX. Comparison of Transfers for Schedule A and B..... 21

IV

LIST OF ILLUSTRATIONS

Figure Page

1. Time Schedules for Experimental Procedures

Employed. 8

2. Procedures for Handling Peritoneal Exudate Cells. 12

CHAPTER I

INTRODUCTION

Delayed hypersensitivity is an allergic state which

develops in response to many microbial agents and contact

substances. These include simple chemicals and materials

from plants such as poison oak and poison ivy (29). In an

effort to work out the mechanism of the delayed response,

workers have made many attempts to passively sensitize

normal animals.

Serum has been reported to be effective when taken

from tuberculous rabbits with well-developed lesions (36).

Normal guinea pigs have been passively sensitized to poison

ivy using serum (9). However, transfers with serum have

only occasionally been successful (3). Other workers used

a serum fraction, which they called an alpha-globulin, to

transfer tuberculin sensitivity (8). Unable to repeat this

transfer, another group said it was not true passive trans-

fer (12).

I The most consistently reproducible method of conferring

i

the passive state of delayed hypersensitivity on normal re-

cipients has been by the injection of leucocytes isolated

from peritoneal exudates, blood, spleen, lung, and lymph

nodes of sensitive donors (1,2,4-7,10-11,13-28,30-35).

Many attempts have been made to use cell extracts instead

of whole cells. These have varied, with many being success-

ful (1,11,13,15-17,25,32-31+) and some unsuccessful

(4,20,21,26,31,35).

One such successful report stressed the importance in

guinea pi-gs of the time lapse between donor sensitization

and passive transfers by cells and their extracts (13).

In that work, it was found that there exists a period when

whole peritoneal exudate cells will passively confer de-

layed sensitivity, but a sonic extract from them will not.

Yet, when the cells were collected four days later, it was

found that both whole cells and sonic extracts would trans-

fer sensitivity.

It would appear that in the cell populations from the

two time periods the "transfer factor" is of different

character or is in a different state. It is possible that

the cells, from the group where extracts do not transfer,

have not yet produced the "transfer factor" when collected.

The group which does yield extract transfers may have al-

ready reached this stage of development. This might be

checked by subjecting these cells to contact with the

specific allergen and then testing their passive transfer

capabilities. This investigation was undertaken to study such

differences in cells collected at two different time in-

tervals after initial sensitization of guinea pigs with

1-fluoro-2,4-dinitrobenzene.

CHAPTER BIBLIOGRAPHY

1. Baram, P. arid Mosko, N. M., "A Dialysable Fraction from Tuberculin Sensitive Human While Blood Cells Capable of Inducing Tuberculin-Delayed Hypersensitivity in Negative Recipients," Immunology, LXXXI (May, 1965), 461-474.

2. Blazkovec, A. A., Sorkin, E. and Turk, J. L., "A Study of the Passive Cellular Transfer of Local Cutaneous Hypersensitivity," International Archives of Allergy and Applied Immunology, XXVII (October, 1965);'

3. Burnet, F. M. and Fenner, F., The Production of Anti-bodies , 2nd ed., New,York, New York, Macmillan,1919.

•4. Chase, M. W., "The Cellular Transfer of Cutaneous Hypersensitivity to Tuberculin," Prodeedings: Society of Experimental Biology and Medicine, LIX (June, 1945), 134-135.

5. Chase, M. W., "The Cellular Transfer of Cutaneous Hypersensitivity," Journal of Bacteriology, LI (May, 1946), 643.

6. Chase, M. W., "Experimental Sensitization with Partic-ular Reference to Picryl Chloride," International Archives of Allergy and Applied Immunology, V (April, 1954),163-1917

7. Chase, M. W., Dameshek, W., Haberman, S., Samter, M., and Sawer, T. L., "The Role .of the Formed Elements of the Blood in Allergy and 'Hypersensitivity," Journal of Allergy, XXVI (June, 1955), 219-252.

8. Cole, L. R. and Favoni?, C. B., "Correlations Between , Plasma Protein Fractions, Antibody Titers and the Passive Transfer of Delayed and Immediate Cutaneous Reactivity to Tuberculin PPD and Tuberculopoly-i saccharides," Journal of Experimental Medicine1, CI (April, 1955), 3 91-470". j

j 9. Crepea, S. B., and Cooke, R. A., "Study on the Mechanism

of Dermatitis Venenata in Guinea Pig with a Demon-stration of Skin-sensitizing Antibody by Passive Transfer," Journal of Allergy, XIX (June, 1948), 353-370.

10. Cummings, M. M., Hoyt, M., and Gottshall, R. Y., "Passive Transfer of Tuberculin Sensitivity in the Guinea Pig," Public Health Reports, LXII (December, 1947), 994-998.

11. Cummings, M. M., Patnode, R. A., and Hudgins, P. C., "Passive Transfer of Tuberculin Hypersensitivity in Guinea Pigs Using Cells Disrupted by Sonic Nibration," The American Review of Tuberculosis and Pulmonary Diseases, LXXIII (February, 1956), 2W-250.

12. Ehrenkranz, N. J. and Waksman, B. H., ^Failure to Trans-fer Tuberculin Sensitivity Passively with Plasma Fractions Containing Alpha-Globulin," Journal of Experimental Medicine, CIV (December, 1956), 93^946.

13. Guthrie, R. K., Lowke, G. E., Ferguson, J. K. and Ellis, W. L., "Contact Hypersensitivity to Simple Chemicals. Time After Donor Sensitization as^a Factor in Passive Transfer," Journal of Investi-gative Dermatology, XLVI (February, 1966), 224-229.

14. Jansen, L. H. and Delden, J. V., "Investigation into the Possibility of Passive Transfer <?f Hypersensitivity to Tuberculin (PPD) in Pigs, and from Man to Pig," Dermatologica, CXXVIIK(February, 1964), 202-222.

15. Jeter, W. S., Tremain, M. M., and Seebohm, P. M., "Passive Transfer of Delayed Hypersensitivity to 2,4-Dinitro-chlorobenzene in Guinea Pigs with Leucocytic Extracts," Proceedings: Society of Experimental Bioiogy and Medicine,LLXXXVI (June, 1954), 251-253.

16. Jeter, W. S., Laurence, K. A., and Seebohm, P. M., "Analysis of Leukocytic Extracts from Guinea Pigs Hypersensitive to Tuberculin and 2,4-Dinitrochloro-henzene," Journal of Bacteriology, LXXIV (November, 1957), 680-683.

17. Kind, P. D., Bocobo, F. C., Curtis, A. C., and Bulala, P., "Cellular Passive Transfer of Contact Hyper-sensitivity to Paraphenylenediamine and to 2,4-Dinitrochlorobenzene in Guinea Pigs," Journal of Investigative Dermatology, XLIV,(January^ 1965), 7-11,

18. Kirchheimer, W. F. and Weiser, R. S., "The Tuberculin Reaction. I. Passive Transfer of Tuberculin Sensi-tivity with Cells of Tuberculous Guinea Pigs," ; Proceedings: Society of Experimental Biology and Medicine, LXVI (OctoberT 1947), 166-17TT

19. Landsteiner, K. and Chase, M. W., "Studies of Animals with Simple Chemical Compounds. VII. Skin Sensitization by Intraperitoneal Injections," Journal of Experimental Medicine, LXXI (February, m o ) , 2i7-2HS.

20. Landsteiner, K. and Chase, M. W., ^Experiments on Transfer of Cutaneous Sensitivity to Simple Compounds," Proceedings: Society of Experi-mental Biology and Medicine, XLIX (April, 1942), 688-690.

21. Landsteiner, K., The Specificity of Serological Reactions, Rev. ed., Cambridge, Massachusetts, Harvard Univer-sity Press, 1947.

22. Lawrence, H. S., "The Cellular Transfer of Cutaneous Hypersensitivity to Tuberculin in Man," Proceedings:

q Society of Experimental Biology and Medicine, LXX1~

23. Lawrence, H. S., "The Cellular Transfer in Humans of De-layed Cutaneous Reactivity to Hemolytic Strepto-cocci," Journal of Immunology, LXVII (February, 1952), 159-178.

24. Lawrence, H. S., "The Transfer in Humans of Delayed Skin Sensitivity to Streptococcal M Substance and to Tuberculin with Disrupted Leucocytes," Journal of Clinical Investigation, XXXIV (February, 1955), 219-230.

25. Lawrence, H. S., "Transfer of Delayed Hypersensitivity to Diptheria Toxin in Man," Journal of Experi-mental Medicine, CIV (September, 195677 321-335.

26. Metaxas, M. N. and Metaxas-Buehler, M., "Passive Trans-fer of Local Cutaneous Hypersensitivity to Tuber-culin," Proceedings: Society of Experimental Biology and Medicine, LXIX (October, 1948), 163-165.

27. Metaxas, M. N. and Metaxas-Buehler, M., "Studies on the Cellular Transfer of Tuberculin Sensitivity in the Guinea Pig," Journal of Immunology, LXXV (November, 1955), 333-347. i

28. Najarian, J. S. and Feldman, J. D., "Specificity of Passively Transferred Delayed Hypersensitivity," Journal of Experimental Medicine, CXVII (September, 1563), 31+1-355. I

29. Raffel, S., "Delayed Hypersensitivities," Progress in Allergy, IV (June, 1954), 173-198.

30. Segre, I), and Sharp, J. B., "Quantitation of Delayed Hypersensitivity in Guinea Pigs by a Local Passive Transfer Reaction," International Archives of Allergy and Applied Immunology, XXVII (March, 1965),

31. Stavitsky, A. B., "Passive Cellular Transfer of the Tuberculin Type of Hypersensitivity," Proceedings: Society of Experimental Biology and Medicine, LXvII (February! 1948), 225-2277

32. Tremaine, M. M. and Jeter, W. S., "Passive Cellular Transfer of Hypersensitivity to Serum Antigens in Rabbits," Journal of Immunology, LXXIV (February, 1955), 96-100.

33. Tsuji, S., Oshima, S., Oshiro, M., and Izumi, T., "Studies on the 'Transfer Factor" of Tuberculin Hypersensi-tivity in Animals," Journal of Immunology, XCIII (November, 1964), 838*849.

34. Turk, J. L., "Passive Transfer of Contact Sensitivity to Picryl Chloride in Guinea Pigs, with Subcellular Material," Nature, CXCI (August, 1961), 915-916.

35. Turk, J. L., "Attempts to Transfer Contact Sensitivity Passively with Subcellular Fractions in the Guinea Pig," International Archives of Allergy and Applied Immunology, XXI (June, 1962), "3T1-325.

36. Zinsser,, H. and Mueller, J. H., "On the Nature of Bac-terial Allergies," Journal of Experimental Medicine, XLI (January, 1925), 159-1777"

CHAPTER II

MATERIALS AND METHODS

Animals

Random bred Hartley strain albino guinea pigs were

obtained from Pine Ridge Caviary, Clinton, Tennessee. Only

animals weighing 350-500 grams were used.

Chemicals

DNFB (l-fluoro-2,^-dinitrobenzene) was obtained from

Matheson Coleman and Bell Chemical Company, Norwood, Ohio.

Light mineral oil (viscosity 80/90) and olive oil were

obtained from E. H. Sargent and Company, Dallas, Texas.

Preparation of Homologous Serum Conjugate

Homologous pooled guinea pig serum was conjugated with

DNFB (U) for use in incubation experiments and testing for

passive systemic anaphylaxis.

Sensitization of Animals

All animals were sensitized by rubbing five drops of two

per cent DNFB in absolute ethyl alcohol into the clippedj neck

using a polished glass rod. This application was repeated

at the same time each day for six days (2).

Skin Testing

Actively sensitzed donor guinea pigs were skin-tested

according to either of two schedules: (A) on the ninth day

after initial contact or (B) on the thirteenth day after

initial contact with DNFB. These schedules are shown in

Figure 1. All recipients were skin-tested for passive sensi-

tization forty-eight hours after injection of transfer prep-

arations. For the skin testing of both donors and recipients,

the hair was clipped from each animal's side and at separate

sites on the bare skin was placed one drop of 0.50 and 0.75

per cent DNFB in olive oil. Each drop of reagent was rubbed

gently into the skin using a separate polished glass rod.

These tests were read after twenty-four hours and were graded

as shown in Table I.

Paint Skin Oil Transfer Skin test Read :ipients skin

tests

1-6

1-6

donors

9 10 13 i

13 m J

17

15

19

16

20

Days for schedule A

Days for schedule B

Fig.l—Time schedules for procedures employed on each dayj.

TABLE I

SKIN TESTS REACTIONS*

Description of Reaction Site Grade

Marked homogenous erythema ++++

Homogenous erythema. +++

Patchy erythema ++

Slight erythema +

Questionable reaction +-

No reaction

J *From reference (2).

Production' and Collection of Peritoneal Exudate

At the same time as the reading of donor skin tests, the

animal's abdomens were shaved and fifteen milliliters of

sterile light mineral oil was injected into the peritoneal

cavity of each in order to evoke a peritoneal exudate (2).

Seventy-two hours after oil injection, animals were exsangui-

nated by intracardial puncture. To obtain cells for passive

transfer, the abdominal wall of each was opened and the cavity

washed three times with Hanks balanced salt solution (3)

modified by the addition of 0.1 per cent gelatin (2). |

Collected cells were sedimented by centrifugation at 2,000

revolutions per minute for twenty minutes. Packed cells jwere

10

washed once in Hanks solution, resedimented at 2,000 revo-

lutions per minute for ten minutes, and the volume recorded.

Washed cells were then resuspended to ten milliliters in Hanks

solution and divided into portions to contain a maximum of

0.75 milliliters packed cells. These cells were handled accord-

ing to one of the following procedures:

A) Cells resuspended in Hanks solution and, without

further treatment, injected into the peritoneal cavity

of a recipient guinea pig.

B) Cells resuspended to a volume of five milliliters in

Hanks solution and placed in an incubator at thirty-seven

degrees centrigrade for thirty minutes. After incubation

the cells were sedimented at 2,000 revolutions per

minute for ten minutes. The supernatant was removed and

injected into the peritoneal cavity of a recipient guinea

pig. The packed cells were washed three times with Hanks

solution 1 and then injected into the peritoneal cavity of

a recipient guinea pig.

C) Cells resuspended to a volume of five milliliters in

Hanks solution and an equal volume of homologous serum

conjugate of DNFB added. This suspension was placed in

an incubator at thirty-seven degrees centrigrade for I

thirty minutes. After incubation the cells were sediment-!

ed at 2,000 revolutions per minute for ten minutes.! The j

supernatant was removed and injected into the peritoneal

cavity of a recipient guinea pig.

11

D) Cells resuspended to a volume of five milliliters

in Hanks solution. These cells were disrupted using the

Branson Sonifier until microscopic examination of samples

showejd:.no;>cells intact. To prevent excessive heating,

all treatment with the sonifier was interrupted at inter-

vals. Containers of cells, while being subjected to

sonic treatment, were immersed in tap water. Suspensions

thus treated were centrifuged for twenty minutes at 2,000

revolutions per minute to sediment solid particles. The

supernatant fluid was decanted and injected into the peri-

toneal cavity of a recipient guinea-pig. The sediment

was washed once in Hanks solution, sedimented for twenty

minutes and the volume recorded. The sediment was then

resuspended and injected into the peritoneal cavity of

a recipient guinea pig.

E) Cells incubated as in (B), the supernatant removed

and injected into the peritoneal cavity of a recipient

guinea pig and the cells washed three times in Hanks

solution. These cells were then disrupted as in (D).

The resulting suspension was sedimented as in (D) and

the supernatant was injected into the peritoneal cavity S

of a recipient guinea pig. The sediment was washed: once

in Hanks solution, the volume recorded and then injected

into the peritoneal cavity of a recipient guinea pig. f

F) Cells incubated as in (C) and then treated as in CE). j

These procedures are summarized in Figure 2.

12

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13

In all incubation experiments, one recipient was injected

with a volume of the homologous serum conjugate equal to that

used in the incubation.

Axillary lymph nodes were removed following harvest of

peritoneal cells and the fat was trimmed away. The nodes were

finely minced with scissors and strained through screen wire

to separate cellss from pulp. Strained cells were washed once

in Hanks solution, sedimented for ten minutes, the volume

recorded, and then resuspended in five milliliters of Hanks

solution for injection into the peritoneal cavity of a

recipient guinea pig (2).

All cells, supernatants, and sediments were injected into

the peritoneal cavity of recipient guinea pigs within five

hours of donor exsanguination.

Testing for Passive Systemic Anaphylaxis

After reading skin tests of recipients, each was injected

intracardially with one milliliter of homologous serum conjugate

containing five milligrams per milliliter serum protein and

observed for symptoms of systemic anaphylaxis (1). Reactions

were graded as shown in Table II. The protein content of the

serum was determined by using the Biuret method (1).

Testing for Presence of Antibody

Incubation supernatants and sonicated cell supernatants

were checked for the presence of antibody using the passive

hemagglutination technique (1).

14

TABLE II

GRADES OF PASSIVE SYSTEMIC ANAPHYLAXIS

Reaction Grade in

Animal

Death

Severe. ++ +

Moderate ++

Slight +

Negative


1. Campbell, D. H., Garey, J. S., Cremer, N. E., and Sussdorf, D. H., Methods In Immunology, New York, New York, W. A. Benjamin, Inc., 1964.

2. Guthrie, R. K., Lowke,̂ G. E., Ferguson, J. K., and Ellis, W. L., "Contact Hypersensitivity to Simple Chemicals. Time After Donor Sensitization as a Factor in Passive Transfer," Journal of Investigative Dermatology, XLVI (February,"~T966) T2H-229.

3. Hanks, J. H., "The Longevity of Chick Tissue Cultures Without Renewal of Medium," Journal of Cellular and Comparative Physiology, XXXI (April, 1948), 235-360.

4. Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Springfield, Illinois, Charles C. Thomas, 1961.

15

CHAPTER III

RESULTS

Skin Tests of Donor Animals

The skin tests of all donor animals showed them to be

sensitive to the chemical used. In Table III are the

reactions of all donors to 0.75 per cent DNFB in olive

oil. Groups XIII-3 and XVII-3 were not skin tested in an

attempt to study the possible effect of skin testing on

passive transfer of the delayed state. These two groups were

run simultaneously with groups XIII-2 and XVII-2.

Skin Tests and Passive Systemic Anaphylaxis

of Recipient Animals

All skin test reactions are to 0.75 per cent and 0.50

per cent DNFB in olive oil. The passive systemic anaphy-

laxis reactions are in response to an intracardial injection

of five milligrams homologous serum protein conjugate of

DNFB contained in a one milliliter dose.

Table IV represents the lymph node recipients' reactions. |

These cells usually gave positive passive transfers of the

delayed state. I i

The results for recipients receiving the peritoneal!

exudate cells which were incubated in Hanks solution are

shown in Table V. The cells from the XIII groups gave passive

16

17

TABLE III

SKIN TEST RESULTS OF DONOR ANIMALS3

Test Number

Number of Donors

Number 4. 4. 4.4.

of Animals with Reactions of + ++ ++ + +_

Group XII1%

1 -r"v

Test 1 10 7 3 0 0 0 0

Test 2 10 8 2 0 0 0 0

Test 3C 10

Test 4 12 12 0 0 0 0 0

Test 5 12 0 4 8 0 0 0

Test 6 24 0 17 7 0 0 0

Group XVIId 1 4

Test 1 10 10 0 0 0 0 0

Test 2 9 9 0 0 0 0 0

Test 3C 10 • *** • • * • • • • •

Test 4 12 12 0 0 0 0 0

Test 5 24 7 8 6 3 0 ;j

-Hi

0

aReactions to 0.75% DNFB in olive oil,

^Donors from schedule A.

^Animals not skin tested.

dDonors from schedule B.

18

TABLE IV

SKIN TEST OF LYMPH NODE RECIPIENTS

Skin Tests Test Cell Volume DNFB Concentration in Olive Oil Number Transferred* 0.75% 0.50%

Group XIII**

Test 1 0.30 4**.; ++ + + 4- + + +

Test 2 0.30 + + + + + + +

Test 3 0.20 + + 4- +

Test 4 0.25 4- -

Test 5

O

rH • O + -

Test 6 0*25 + + + + +

Group XVII***

Test 1 0.30 + 4* + + 4- + 4-

Test 2 0.10 + +4 +

Test 3 0.10 4- + ~

Test •+ 0.35 + 4-

Test 5 0.35 + - -

*Cell volume in milliliters.

**Animals from schedule A.

*fc*Animals from schedule B.

19

TABLE V

SKIN TESTS AND PASSIVE SYSTEMIC ANAPHYLAXIS OF RECIPIENTS RECEIVING PERITONEAL EXUDATE CELLS

AFTER INCUBATION IN HANKS

Skin Tests Passive Test

Number Cell Volume Transferreda

DNFB Concentration in Olive Oil 0.75% 0.50%

Systemic Anaphylaxis

Group XIIIb

Test 1 0.75 ++ +

Test 2 0.75 + + + -

Test 3 0.75 + + -

Test 4 0.75 ++ + -

Test 5 0.75 + + - -

Test 6 0.75 + + - -

Group XVIT0

Test 1 0.75 + -

Test 2 0.60 + - - -

Test 3 0.75 - - -

Test H 0.75 + - + - -

Test 5 0.75 + - -

a Cell volume in milliliters,

b Animals from schedule A.

c Animals from schedule B.

20

transfers after the incubation, while the cells from the

XVII groups never did. The recipients of the supernatants

after these incubations gave the reactions shown in Table

VI. These supernatants conferred activity to all the

recipients of both time periods.

When peritoneal exudate cells were incubated with

homologous conjugated serum, the recipients of such cells

gave the reactions in Table VII. Cells from XIII groups

gave no reactions. However, cells from XVII groups gave

positive passive transfers of the delayed state and passive

systemic anaphylaxis. When the supernatants from these

incubations frere injected, the recipients from the XVII

groups were completely negative. However, the XIII groups

exhibited both the delayed response and passive systemic

anaphylaxis. This is shown in Table VIII.

Two experiments, XIII-6 and XVII-5, were run to study

all procedures which were described in the preceding chapter

for passive transfers. Table IX shows the differences and

similarities of the passive transfer capabilities of the two

time periods studied.

The control animals, which received homologous serum !

conjugate equal in volume to that used in the incubation j

part of the experiments, were all negative. |

Passive Hemagglutination Test

Using the passive hemagglutination technique, all

supernatants from incubation experiments in groups XIII-5;,

21

TABLE VI

SKIN TESTS AND PASSIVE SYSTEMIC ANAPHYLAXIS OF RECIPIENTS RECEIVING SUPERNATANT AFTER INCUBATION IN HANKS OF

PERITONEAL EXUDATE CELLS

Skin Tests Passive Test Number

DNFB Concentration in Olive Oil 0.75% 1 0.50%


Group XIIIa

Test 4 +++ -

Test 5 ++ -

Test 6ab + + -

Test 6b \

+ + -

Group XVIIc

Test 3 + + + -

Test 4 + + + -

Test 5a^ + -

Test 5b + +-

—L

-

aAnimals from schedule A.

^XIII-6 Ifcun in duplicate,


XVII-5 rvm in duplicate.

22

TABLE VII

SKIN TESTS AND PASSIVE SYSTEMIC ANAPHYLAXIS OF RECIPIENTS RECEIVING PERITONEAL EXUDATE CELLS AFTER

INCUBATION WITH SERUM CONJUGATE


- Cell Volume Transferred



Group XIIIb

Test 1 0.75

Test 2 0.75 _ -

Test 3 0.75 - -

Test 4 0.75 - -

Test 5 0.75 - -

Test 6 0.75 - -

Group XVIIC

Test 1 0.75 ++ + +

Test 2 0.60 ++ + +

Test 3 0.75 ++ + +++

Test 4 0.75 + +

Test 5 0.75 + +- +

a Cell volume in milliliters,

b Animals from schedule A.


23

TABLE VIII

SKIN TESTS AND PASSIVE SYSTEMIC ANAPHYLAXIS OF RECIPIENTS RECEIVING SUPERNATANT AFTER INCUBATION OF PERITONEAL

EXUDATE CELLS WITH SERUM CONJUGATE




Group XIII*

Test 1 + + + 4-

Test 2 + +

Test 3 + - -

Test 4 + - +

Test 5 + - -

Test 6 + - ++

Group XVII**

Test. 1 + - + - -

Test 2 - - -

Test 3 - - -

Test k + - + - -

Test 5 — -| |

*Animals from schedule A.

**Animals from schedule B,

24

TABLE IX

COMPARISON OF TRANSFERS FOR SCHEDULES A AND B

Procedure Represented Skin Tests


Passive Systemic

Anaphylaxis

Experiment XIII-6a

(A) Whole Celjlsc

(D) Sonic Supernatant (D) Sonic Sediment (B) Whole Cells (B) Incubation Supernatant (E) Sonic Supernatant (E) Sonic Sediment (C) Whole Cellse

(C) Incubation Supernatant (F) Sonic Supernatant (F) Sonic Sediment

+ +

+-+

++

Experiment XVII-5^3

(A) Whole Cells0

(D) Sonic Supernatant (D) Sonic Sediment (B) Whole Cellsd

(B) Incubation Supernatant (E) Sonic Supernatant (E) Sonic Sediment (C) Whole Cellse

(C) Incubation Supernatant (F) Sonic Supernatant (F) Sonic Sediment

+ +

+

+

+

+

+ -

^From schedule A.

^From schedule B. j i

cPeritoneal exudate cells untreated* ; !

^Peritoneal exudate cells incubated in Hanks. |

ePeritoneal exudate cells incubated with serum conjugate.

25

XIII-6, XVII-1*, and XVII-5 and all sonicated cell supernatants

from groups XIII-6 and XVII-5 were tested for presence

of antibody. In no test was a positive hemagglutination

observed.

CHAPTER IV

DISCUSSION

These experiments have shown that there are differences

in the reactivity of peritoneal exudate cells collected at

different time periods. As reported earlier (5), it was

found that sonic extracts from peritoneal exudate cells

collected thirteen days after donors' sensitization would

not passively transfer the delayed state, although whole

cells did. If the cells were collected four days later,

both sonic extracts and whole cells gave positive passive

transfers. Many workers have reported positive transfers

using sonic extracts (1,3,5,6,7,8,9,12,11,18,19,20), while

others have been unsuccessful (2,10,11,15,17,21). These

discrepancies may have been in the animal species used,

the allergens used, the cell types used, time schedules, or

other peculiarities of each experiment.

As reported in recent reviews (4,13,16),it was found

that lymph node cells from sensitive donors will passively i

transfer delayed sensitivity. j

In the experiments where the cells were incubated with 1

homologous serum conjugate there is more evidence that the

cells of the two time periods are different. These results,

Tables VII and VIII, show that the XIII cells, from the time !

period which does not yield a sonic extract that will passively |

26 !

27

transfer, are desensitized by contact with homologous serum

conjugate. Some of the transfer activity did appear in the

supernatant after incubation. The XVII cells, collected

four days later and which yield a positive passive trans-

ferring sonic extract, seem to have the transfer activity

"fixed" with them. No transfer activity appeared in the

supernatant after incubation. Incubation experiments

have been devised by others whereby collected cells were

allowed to come in contact with the specific allergen

in vitro (12,13). It is not possible at this time to

directly compare these results with others for, according

to Chase (16), this incubation for contact sensitivity

in the guinea pig has not been reported.

A technique designed at first to be a control over

the preceeding experiments developed into still another

experimental procedure which demonstrated the afore-

stated time differences in collected cells. When cells

were incubated in Hanks solution, it was found that both

groups release transfer activity into the supernatant as

shown in Table VI. However, these results show that the

cells which did not yield sonic extract transfers would

still yield whole cell transfers. And the cells which did

yield sonic extract transfers no longer gave whole cell.-1 I

transfers. This type of cell incubation has not beenrre-I

ported previously. In working with human leucocytes, j

Lawrence (12) found that incubation of cells in serum for a

short period would yield a supernatant which gave passive

transfers-. I

28

In the time periods studied, it was found that passive

systemic anaphylaxis did not become evident in recipients

unless the cells were first incubated in the presence of

homologous serum conjugate. After such incubation, in the

group which gave no sonic extract transfers, only the super-

natant gave positive passive systemic anaphylaxis. It

appears that, like the delayed properties, the anaphylactic

property of these cells is released in the presence of the

conjugate. In the group which gave sonic extract transfers

the cells retained their anaphylactic property after con-

tact or were stimulated to produce it by the contact. It

may be that these reactions are not due to classical anti-

body. This is indicated by the negative hemagglutination

tests for the samples yielding positive passive systemic

anaphylaxis. Perhaps these positive reactions were due to an

antibody other than the classical type.

Unexplained by these experiments is the apparent in-

compatibility in the failure of sonic extracts of cells

collected thirteen days after donor sensitization to give

passive transfers»while incubation of these cells yields a

supernatant which will transfer. This might be explained i

by the experiments of Tsuji et al. (18), in which they found

that an "inhibitor" was indicated and could be removed from

cellular extracts by dialysis. If this is the case, then

the "inhibitor" is either not present in the XVII cells or

is in low enough concentration in proportion to the "trans-

fer factor" that it cannot express itself in these sonic!

29

extracts. Perhaps the XVII cells, Table V, which have re-

leased "transfer factor", do not give positive transfers

after Hanks incubation because the "inhibitor" is now in

sufficient quantity to mask it.

The experiments which have been discussed support the

idea (5) of differences in cellular reactivities at differ-

ent time periods in guinea pigs hypersensitive to 1-fluoro-

2, if-dinitrobenzene.


1. Baram, P. and Mosko, N. M., "A Dialysable Fraction from Tuberculin Sensitive Human White Blood Cells Capable of Inducing Tuberculin-Delayed Hypersensi-tivity in Negative Recipients," Immunology, LXXXI (May, 1965), 461-474.

2. Chase, M. W., "The Cellular Transfer of Cutaneous Hypersensitivity to Tuberculin," Proceedings: Society of Experimental Biology and Medicine, LIX (June, 1945), 134-133".

3. Ehrei^kranz, N. J. and Waksman, B. H., "Failure to Transfer "Tuberculin Sensitivity Passively with Plasma Fractions Containing Alpha Globulin," Journal of Experimental Medicine, CIV (December, 1956), 9 35-946.

4. Gowans, J. L. and Mc Gregor, D. D., "The Immunological Activities of Lymphocytes," Progress in Allergy, IX (June, 1965), 1-63.

5. Guthrie, R. K., Lowke, G. E., Ferguson, J. K., and Ellis, W. L., "Contact Hypersensitivity to Simple Chemicals. Time After Donor Sensitization as a Factor in Passive Transfer," Journal of Investigative Dermatology, XLVI, (February, 1966), 224-229.

6. Jansen, L. H. and Delden, J. V., "Investigation into the Possibility of Passive Transfer of Hypersensitivity to Tuberculin (PPD) in Pigs, and from Man to Pig," Dermatologica , CXXVIII (February, 1964), 202-222.

7. Jeter, W. S., Lawrence, K. A. and Seebohm, P. M., "Analysis of Leukocytic Extracts from Guinea Pigs Hypersensitive to Tuberculin and 2,4-Dinitrochloro-benzene," Journal of Bacteriology, LXXIV (November, 1957), 680-683.

8. Kind, P. D., Bocobo, F. C., Curtis, A. C., and Bulala, P., "Cellular Passive Transfer of Contact Hypersensi-tivity to Paraplenylenediamine and to 2,4-Dinitro-chlorobenzene in Guinea Pigs," Journal of Investi-gative Dermatology, XLIV (January, 196577 7-11.

30

31

9. Kirchheimer, W. F. and Weiser, R. S., "The Tuberculin Reaction. I. Passive Transfer of Tuberculin Sensitivity with Cells of Tuberculous Guinea Pigs," Proceedings; Society of Experimental Biology and Medicine, LXVl (["October, 1947), 166-170.

10. Landsteiner, K., The Specificity of Serological Reactions, Rev. ed., Cambridge, Massachusetts, Harvard University Press, 1947.

11. Lawrence, H. S., "The Cellular Transfer of Cutaneous Hypersensitivity to Tuberculin in Man," Proceedings: Society of Experimental Biology and Medicine, LXXI, (August ,~~T949), 516-522.

12. Lawrence, H. S. and Pappenheimer, A. M., "Transfer of Delayed Hypersensitivity to Diptheria Toxin in Man," Journal of Experimental Medicine, CIV (September, 1956T,321-335 .

13. Lawrence, H. S., Cellular and Humoral Aspects of the Hypersensitive States, New York, New York, Paul B. Hoeber, Inc.7 19 59.

14. Metaxas, M. N. and Metaxas-Buhler, M., "Passive Transfer of Local Cutaneous Hypersensitivity to Tuberculin," Proceedings: Society of Experimentali-Biology and Medicine, EXIX (October, 1948) , 163-165.

15. Metaxas, M. N. and Metaxas-Buhler, M., "Studies on the Cellular Transfer of Tuberculin Sensitivity in the Guinea Pig," Journal of Immunology, LXXV (November, 1955), 333-347::

16. Raffel, S., ed., The Medical Clinics of North America, Philadelphia, Pennsylvania, W. B. Saunders Company, 19 65.

17. Tremaine, M. M. and Jeter, W. S., "Passive Cellular Transfer of Hypersensitivity to Serum Antigens in Rabbits," Journal of Immunology, LXXIV (February, 1955), 96-100. i

18. Tsuji, S., Oshima, S., Oshiro, M. and Izumi, T., "Studies on the'Transfer Factor' of Tuberculin Hypersensi-tivity in Animals," Journal of Immunology, XCIII (November, 1964), 838-849.

32

19. Turk, J. L., "Passive Transfer of Contact Sensitivity to Picryl Chloride in Guinea Pigs, with Sub-cellular Material, " Nature, CXCI (August, 1961), 915-916.

20. Turk, J. L. and Asherson, G. L., "Attempts to Transfer Contact Sensitivity Passively with Subcellular Fractions in the Guinea Pig," International Archives of Allergy and Applied Immunology, XXI (June, 1962), 321-325.

21. Zinsser, H. and Mueller, J. H., "On the Nature of Bac-terial Allergies," Journal of Experimental Medicine, XLI (January," 19 3"5T7 159-177.

CHAPTER V

SUMMARY

Guinea pigs were sensitized with l-fluoro-2,4-dinitro-

benzene. Peritoneal exudate cells were studied in two

time periods in relation to time lapse after donor sensiti-

zation. Cells collected thirteen days after sensitization

differed from those collected seventeen days after sensiti-

zation in terms of transfer reactivities. Experiments

showed the following:

1. Sonic extracts of thirteen day cells failed to

transfer while seventeen day sonic extracts did transfer.

2. Incubation in Hanks gave rise to a release into the

mediumsa transfer reactivity. The thirteen day cells

were still capable of positive transfers, but the seven-

teen day cells no longer had this capability.

3. Incubation with homologous serum conjugate desensi-

tized the thirteen day cells, leaving only the supernatant

with transfer activity. Seventeen day cells retained

transfer activity, with none appearing in the supernatant.

Passive systemic anaphylaxis was transferred by: the

supernatant from the incubation of thirteen day cells

with homologous serum conjugate. Such incubation of

seventeen day cells left them, but not the supernatant,

with the transfer capability. | i

33 - I

3H

Only after incubation of cells with homologous serum

conjugate was passive systemic anaphylaxis observed in re-

cipient guinea pigs. Passive hemagglutination tests of all

incubation and sonication supernatants failed to demonstrate

classical antibody.

It remains to be determined whether an "inhibitor" of

"transfer factor" is really present in the time periods

studied and, if so, the mechanism of its action.

BIBLIOGRAPHY

BOOKS

Burnet, F. M. and Fenner, F., The Production of Antibodies, 2nd ed., New York, New York, Macmillan,-T949.

Campbell, D. H., Garey, J. S., Cremer, N. E., and Sussdorf, D. H., Methods in Immunology, New York, New York,

Landsteiner, K., The Specificity of Serological Reactions. Rev. ed. Cambridge, Massachusetts, Harvard University Press, 1947.

Lawrence, D. H., Cellular and Humoral Aspects of the Hyper-sensitive States, New York, New York, Paul B. Hoeber, Inc., 19591

Raffel, S., The Medical Clinics of North America, Philadelphia, Pennsylvania, W. B~ Saunders Company, iabt>.

ARTICLES

Baram, P. and Mosko, N. M., "A Dialysable Fraction from Tuberculin Sensitive Human White Blood Cells Capable of Inducing Tuberculin-Delayed Hypersensitivity in Negative Recipients," Immunology LXXXI (May, 1965), 461-474.

Blazkovec, A. A., Sorkin, E. and Turk, J. L., "A Study of the Passive Cellular Transfer of Local Cutaneous Hyper-sensitivity," International Archives of Allergy and Applied Immunology, XXVlI (October, I9F5")̂ 2 8 a - 3 0"3T~

Chase, M. W., "The Cellular Transfer of Cutaneous Hypersen-sitivity," Journal of Bacteriology, LI (May, 1946), 643.

Chase, M. W., "The Cellular Transfer of Cutaneous Hyper-sensitivity to Tuberculin," Proceedings; Society of Experimental Biology and Medicine, LIX (June, 1945), 134-135.

cha.'-se ; Chase, M. W., "Experimental Sensitization with Particular

Reference to Picryl Chloride," International Archives of Allergy and Applied Immunology, V (April, li)b4), 163-191.

35

36

Chase, M. W. , Dameshek, W. , Haverman, S., Samter, M. and Sqiver, T. L., "The Role of the Formed Elements of the Blood in Allergy and Hypersensitivity," Journal of Allergy, XXVI (June, 1955), 219-252.

Cole, L. R. and Favour, C. B., "Correlations Between Plasma Protein Fractions, Antibody Titers, and the Passive Transfer of Delayed and Immediate Cutaneous Reactivity to Tuberculin PPD and Tuberculopolysaccharides," Journal of Experimental Medicine, CI (April, 1955), 391-420.

Crepea, S. B. and Cooke, R. A., "Study on the Mechanism of Dermatitis Venenataiin Guinea Pigs with a Demonstration of Skin-sensitizing Antibody by Passive Transfer," Journal of Allergy, XIX (June, 1948), 353-370.

Cummings, M. M., Hoyt, M. and Gottshall, R. Y., "Passive. Transfer of Tuberculin Sensitivity in the Guinea Pig," Public Health Reports, LXII (December, 1947), 994—998.

Cummings, M. M., Patnode, R. A., and Hudgins, P. C., "Passive Transfer of Tuberculin Hypersensitivity in Guinea figs Using Cells Disrupted by Sonic Vibration,V The American Review of Tuberculosis and Pulmonary Diseases, LXSTXT (February, 195677 246-250.

Ehrenkranz, N. J.5and Waksman, B. H., "Failure to Transfer Tuberculin Sensitivity Passively with Plasma Fractions Containing Alpha Globulin," Journal of Experimental Medicine, CIV (December-* 1956Tj 935-941".

Gowans, J. L. and Mc Gregor, P. P., "The Immunological Activities of Lymphocytes," Progress in Allergy, IX (June, 1965), 1-63.

Guthrie, R. K., Lowke, G. E., Ferguson, J. K., and Ellis, W. L. , "Contact Hypersensitivity to Simple Chemicals. Time After Donor Sensitization as a Factor in Passive Transfer," Journal of Investigative Dermatology, XLVI (February, 19 6677 224-229.

Hanks, J. H., "The Longevity of Chick Tissue Cultures Without Renewal of Medium," Journal of Cellular and Comparative Physiology, XXXI (April, 1941*7, 235-260. i

Jansen, L. H. and Delden, J. V., "Investigation into the ! Possibility of Passive Transfer of Hypersensitivity to Tuberculin (PPD) in Pigs, and From Man to Pig," ! Dermatologica, CXXVII (February, 1964), 202-222. :

37

Jeter, W. S., Tremaine, M. H., and Seebohm, P. M., "Passive Transfer of Delayed Hypersensitivity to 2,4-Dinitro-chlorobenzene in Guinea Pigs with Leucocytic Extracts," Proceedings: Society of Experimental Biology and Medicine,LXXXVI (June," 1954), 251-253.

Jeter, W. S. and Laurence,K. A. and Seebohm, P. M., "Analysis of Leukocytic Extracts from Guinea Pigs Hypersensitive to Tuberculin and 2,4-Dinitrochlorobenzen," Journal of Bacteriology, LXXVI (November, 1957), 680-683.

Kind, P. D. , Bocobo, F. C., Curtis, A. C., and Bulala, P., "Cellular Passive Transfer of Contact Hypersensitivity to Paraphenylenediamine and to 2,4-Dinitrochlorobenzene in Guinea Pigs," Journal of Investigative Dermatology, XLIV (January, 1965)", 7-11.

Kirchheimer, W. F. and Weiser, R. S., "The Tuberculin Reaction. I. Passive Transfer of Tuberculin Sensi-tivity with cells of Tuberculous Guinea Pigs," Proceedings: Society of Experimental Biology and Medicine, LXVI (October, 1947), 166-170.

Landsteiner, K. and Chase, M. W., "Experiments on Transfer of Cutaneous Sensitivity to Simple Compounds," Proceedings: Society of Experimental Biology and Medicine, XLIX (April, "^9 4 2), 688-690 .

Landsteiner, K. and Chase, M. W., "Studies on the Sensitization of Animals with Simple Chemical Compounds. XII. Skin Sensitization by Intraperitoneal Injections," Journal of Experimental Medicine, LXXI (February, 1940), 237-245.

Lawrence, H. S., "The Cellular Transfer in .Humans of Delayed Cutaneous Reactivity to Hemolytic Streptococci," Journal of Immunology, LXVIII (February, 1952), 159-178.

Lawrence, H. S., "The Cellular Transfer of Cutanous Hyper-sensitivity to Tuberculin in Man," Proceedings: Society of Experimental Biology and Medicine, LXXI

~~*"(August, 1949; , 516-522.

Lawrence, H. S., "The Transfer in Humans of Delayed Skin j Sensitivity to Streptococcol M Substance and to Tuberculin with Disrupted Leucocytes," Journal of Clinical Investigation, XXXIV (February, 1955), 7T9-230.

Lawrence, H. S. and Pappenheimer, A. M., "Transfer of Delayed Hypersensitivity to Diptheria Toxin in Man," Journal of Experimental Medicine, CIV (September, 1956), 321-335.

38

Metaxas, M. N. and Metaxas-Buhler, H., "Passive Transfer of Local Cutaneous Hypersensitivity to Tuberculin," Proceedings: Society of Experimental Biology and Medicine, EXIX (October, 194-8), 163-1651

Metaxas, M. N. and Metaxas-Buehler, M., "Studies on the Cellular Transfer of Tuberculin Sensitivity in the Guinea Pig," Journal of Immunology, LXXV (November, 1955), 333-347:

Najarian, J. S. and Feldman, J. D., "Specificity of Passively Transferred Delayed Hypersensitivity," Journal of Experimental Medicine, CXVIII (September^ 1963), m-3b2.

Raffel, S., "Delayed Hypersensitivities," Progress in Allergy. IV (June, 1951-0 , 173-178.

Segre, D. and Sharp, J. B., "Quantitation of Delayed Hypersensitivity in Guinea Pigs by a Local Passive Transfer Reaction," International Archives of Allergy and Applied Immunology, XXVII (March, 1965), 82-101.

Stavitsky, A. B., "Passive Cellular Transfer of the Tuber-culin Type of Hypersensitivity," Proceedings: Society of Experimental Biology and Medicine, LXVII (February, m s ) ,

Tremaine, M. M. and Jeter, W. S., "Passive Cellular Transfer of Hypersensitivity to Serum Antigens in Rabbits," Journal of Immunology, LXXXIV (February, 1955), 96-100.

Tsuji, S., Oshima, S., Oshiro, M., and Izumi, T., "Studies on the 'Transfer Factor1 of Tuberculin Hypersensitivity in Animals," Journal of Immunology, XCIII (November, 1964), 83 8-8491

Turk, J. L., "Passive Transfer of Contact Sensitivity to Picryl Chloride in Guinea Pigs, with Subcellular Material," Nature, CXCI (August, 1961), 915-916.

Turk, J. L., "Attempts to Transfer Contact Sensitivity Passively with Subcellular Fractions in the Guinea Pig," International Archives of Allergy and Applied Immuno-logy, XXlTjune, 1962), 321-325 . ~~

Zinsser, H. and Mueller, J. H., "On the Nature of Bacterial Allergies," Journal of Experimental Medicine, XLI (January, 1925)159-T77.

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