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Ocular findings in systemic cutaneous

basophil hypersensitivity
Mitchell H. Friedlaender and Richard Cyr

SCBH was induced by immunizing guinea pigs with a protein antigen and challenging 1 week
later with a large intraperitoneal dose of the same antigen. Animals developed a delayed-onset,
erythematotts skin rash and dermal infiltration by basophils and eosinophils. The uveal tracts
of these animals were infiltrated by eosinophils, as were several other internal organs. The eye
is affected in SCBH, and as in other forms of ocular cell-mediated hypersensitivity, the
eosinophil is a prominent cellular component of these reactions.

Key words: delayed hypersensitivity, cutaneous basophil hypersensitivity,


eosinophil, basophil, uveal tract, systemic cutaneous basophil hypersensitivity

T wo types of delayed cutaneous hypersen-


sitivity reactions can be distinguished in ani-
lymphocytes. However, DH is a long-lasting
state of hypersensitivity effected by lympho-
mals and man with the use of clinical and cytes with relatively high avidity for antigen,
microscopic criteria.1"3 "Classic" delayed whereas CBH is often a transient state of hy-
hypersensitivity (DH) is favored when guinea persensitivity mediated by lymphocytes of
pigs are immunized with antigens in myco- lower average avidity.
bacteria-containing adjuvants. DH skin reac- Studies of cell-mediated hypersensitivity
tions are erythematous, indurated, and infil- reactions in guinea pig eyes have also differ-
trated by large numbers of mononuclear entiated two types of ocular responses which
cells. Cutaneous basophil hypersensitivity correspond to DH and CBH in the skin.4' 5
(CBH) is induced when mycobacteria-con- Animals primed for DH develop highly cellu-
taining adjuvants are not used during im- lar reactions in their corneas and uveal tracts
munization, and these skin reactions are typ- which contain large numbers of mononuclear
ically erythematous and nonindurated and cells. Those primed for CBH develop corneal
contain large numbers of basophilic leuko- reactions containing large numbers of baso-
cytes in addition to lymphocytes. Both DH phils, but the limbus and uveal tracts of these
and CBH reactions are delayed in onset for animals are heavily infiltrated by eosinophils.
several hours after challenge, and both types To further evaluate the ocular features of
of reactions are mediated by sensitized T these reactions, we studied the eyes of
guinea pigs primed for systemic cutaneous
basophil hypersensitivity (SCBH), a systemic
From the Francis I. Proctor Foundation for Research in
Ophthalmology, University of California, San Fran-
expression of cell-mediated hypersensitivity
cisco, Calif. in which guinea pigs are immunized for CBH
Supported by National Institutes of Health Research and challenged intraperitoneally with a large
Grants EY-02502 and EY-01597, and National Insti- dose of antigen.6
tutes of Health Fellowship Grant EY-05086.
Submitted for publication Dec. 4, 1978. Materials and methods
Reprint requests: Dr. Mitchell H. Friedlaender, Francis
I. Proctor Foundation, University of California, San Immunization and elicitation of SCBH. Thirty
Francisco, Calif. 94143. male and female Hartley strain guinea pigs, 400 to

964 0146-0404/79/090964 +06$00.60/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc.

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Volume 18
Number 9 Ocular findings in SCBH 965

Fig. 1. Delayed onset, erythematous rash elicited 24 hr after intraperitoneal challenge of


guinea pig primed for SCBH with OA.

Table I. Differential counts of cells infiltrating guinea pig uveal tract


in systemic delayed hypersensitivity
Time after No. of
challenge (hr) eyes Mononuclears Neutrophils Eosinophils Basophils

24 § 78.2 ± 8.0* 3.7 ± 2.7 18.2 ± 4.1


m M 83.0 ± 8.5 5.9 ± 5.2 11.1 ± 1.4
72 10 85.3 ± 2.6 3.2 ± 1.1 12,0 ± 2.3
'Mean percent of cells counted ± S.E.M.

600 gm, were immunized in the footpads with 100 buffer, pH 7.4, or else placed in Helly's fluid ac-
/Ltg of either ovalbumin (OA) or bovine gamma cording to the protocol of Askenase.8 The tissues
globulin (BGG), both obtained from Miles Labora- fixed by the former method were embedded in
tories, Elkhart, Ind., in 0.1 ml of incomplete JB-4 plastic embedding medium (Polysciences,
Freund's adjuvant (IFA) (Difco Laboratories, De- Warrington, Pa.), sectioned at 1 fim, and stained
troit, Mich.). Five to 7 days later, SCBH was in- with Giemsa's reagent (Fisher Scientific, Pitts-
duced by an intraperitoneal challenge injection of burgh, Pa.). Those fixed in Helly's fluid were em-
50 mg of the antigen used for immunization in 2 ml bedded in paraffin, sectioned at 5 /im, and stained
of saline. Animals were observed at time intervals with Giemsa's reagent. A certain number of tis-
ranging from 1 to 72 hr after challenge and sac- sues were fixed initially in 1.5% glutaraldehyde
rificed at 24, 48, or 72 hr. Skin reactions were prior to transferring them to Karnovsky's fixative
more readily observed in animals after shaving and and processing them for JB-4 embedding. Differ-
depilating with Nair (Carter-Wallace Inc., Cran- ential cell counts were performed on 1 /jun sec-
bury, N, J.)? but depilation itself did not affect the tions of uveal tract by means of a method previ-
development of SCBH reactions. ously described. 4 ' 5 Mean percentages ± the
Histology. Eyes and other tissues were re- standard error of the mean were calculated for
moved from animals sacrificed with pentobarbital several eyes removed at 24, 48, and 72 hr time
anesthesia and immediately placed in fixative. Tis- intervals (Table I).
sues were either fixed in Karnovsky's mixture of Controls. The eyes and other organs of several
glutaraldehyde and paraformaldehyde7 for 5 hr control animals were examined microscopically
and then transferred to 0.1M sodium cacodylate with both methods of tissue processing. Control

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Invest. Ophthalmol. Visual Set.
966 Friedlaender and Cyr September 1979

whereas only two animals had no perceptible


rash at the time of sacrifice.
Histology of SCBH skin reactions. The
papillary and reticular dermis of the ery-
thematous skin reactions were infiltrated by
moderate numbers of inflammatory cells. A
consistent finding at 24, 48, and 72 hr was the
presence of both mononuclear cells and neu-
trophils. In general, larger numbers of neu-
trophils were observed at 24 hr, whereas
mononuclear cells predominated at 48 and 72
hr. Eosinophils and basophils (Fig. 2) were
consistently found at all time intervals. In
some tissue sections, especially those taken
at 48 and 72 hr, eosinophils were more prom-
inent than basophils. The number of baso-
phils and eosinophils varied considerably in
skin taken from different animals, and in
some sections only an occasional eosinophil
or basophil could be identified among a back-
ground of mononuclear cells and neutrophils.
Fig. 2. Basophils (b) infiltrating the dermis of In other skin reactions, eosinophils and
guinea pig primed for SCBH. (Gienisa; X1080.) basophils were seen throughout the dermis
and often appeared in collections within and
animals were treated in one of the following three outside of vessels.
ways: (1) not sensitized, (2) sensitized but not chal- The venules of the upper dermis were
lenged, (3) challenged intraperitoneally but not often dilated and compacted with large num-
sensitized. bers of erythrocytes and leukocytes, many of
which were basophils and eosinophils. Fixed-
Results tissue mast cells were observed throughout
Appearance of SCBH reactions. Guinea the dermis. They were easily distinguishable
pigs primed for SCBH with either OA or from basophils because of their round nuclei
BGG and challenged 5 to 7 days later with an and small, darkly staining granules. Baso-
intraperitoneal injection of the same antigen phils, which are granulocytes, have seg-
used for sensitization developed a delayed- mented nuclei and large, oval or pear-shaped
onset, erythematous, macular, and very granules. Both basophils and mast cells ap-
slightly papular rash (Fig. 1). The rash was peared intact and did not show evidence of
detectable 6 hr after challenge but was best degranulation. Little or no edema was ob-
observed at 24 hr and was somewhat faded by served in the skin, and the deeper dermis
48 hr. The rash began on the flanks and nape and subcutis showed little or no inflam-
of the neck and then extended to the abdo- mation.
men, legs, and back. Initially, macular skin Histology of eye reactions. Ocular inflam-
lesions were discrete but later became con- mation was far more prominent in the cho-
fluent. The eyelids were often diffusely roid and ciliary body than in any other area of
erythematous, especially when the rash was the eye. The most striking finding was the
severe and diffuse. The conjunctivas were large number of eosinophils which infiltrated
often slightly injected; however, this was not the uveal tract (Fig. 3). Unlike the skin,
a consistent finding and could not be corre- basophils were either totally absent or only
lated with the severity of the rash. Of the 30 rarely observed. Eosinophils were seen
animals primed for SCBH, 28 developed a throughout the choroid and ciliary body but
diffuse rash involving all areas of the skin, seemed to have a predilection for the outer

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Volume 18
Number 9 Ocular findings in SCBH 967

# - " * • • !

Fig. 3. Eosinophils (e) infiltrating the uveal tract of guinea pig primed for SCBH. (Giemsa;
X1080.)

layers of the choroid, where they were fre- of the total cell counts. Eosinophils were
quently lined up near the sclera. Eosinophils numerous at all three time intervals, account-
were sometimes observed within choroidal ing for 18% of the infiltrate at 24 hr, 11% at
blood vessels, especially in eyes removed at 48 hr, and 12% at 72 hr. Neutrophils were
24 hr. The number of eosinophils observed observed in relatively small numbers at all
correlated with the intensity of the overall three time intervals (3% to 6%), and no
uveal inflammation. Large numbers of eosin- basophils were seen in the uveal tract for any
ophils and other inflammatory cells were of the cell counts. The counts for any one cell
present at 24, 48, and 72 hr. type did not change significantly during the
Mononuclear cells (mainly lymphocytes and three time intervals according to Student's t
lymphoblasts) were observed in all sections of test (p < 0.05).
the uveal tract and were the predominant cell The conjunctiva showed only mild evi-
at all three time intervals. Mast cells were dence of inflammation microscopically. An
present in large numbers throughout the occasional eosinophil, neutrophil, or mono-
uveal tract and, as in the skin, showed no nuclear cell could however, often be found,
evidence of degranulation. Choroidal blood The cornea, limbus, and other areas of the
vessels were often filled with large numbers eye were free of inflammatory cell infiltration
of erythrocytes and leukocytes (especially or other pathologic changes in all animals.
eosinophils and neutrophils). Endothelial cell Histology of other tissues. Eosinophils
hypertrophy was sometimes observed; how- were prominent in the microvasculature of
ever, this was not as striking as the endothe- the internal organs; however, they were
lial changes seen when sensitized animals are found less consistently in lung, thymus,
challenged with ocular injections of protein spleen, and lymph nodes than in the uveal
antigens.4' 5 In previous studies,4 auto- tract. Some sections of lung contained large
radiography was used to demonstrate endo- numbers of eosinophils, but since these cells
thelial cell hyperplasia; however, such stud- are sometimes found in normal guinea pig
ies were not performed in the present series lung,6 their significance is uncertain. Eosino-
of experiments. phils were found in the red pulp of spleen
Differential counts of cells infiltrating the and in the medulla of the thymus and lymph
uveal tract (Table I) indicated that mononu- nodes. Although eosinophils in these organs
clear cells were the most numerous inflam- were most consistently observed in animals
matory cell at 24, 48, and 72 hr after intra- sacrificed 24 hr after challenge, they were
peritoneal challenge, comprising 78% to 85% also seen in tissues removed at 48 and 72 hr.

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Invest. Ophthalmol. Visual Sci.
968 Friedlaender and Cyr September 1979

Controls. The three groups of control ani- phils seem to have a predilection for the
mals lacked hyperacute inflammatory cell outer layers of the choroid, a variation which
infiltration of the uveal tract. Fixed tissue may be species-related and perhaps deter-
mast cells were consistently seen in large mined by different patterns of uveal blood
numbers throughout the uveal tract (espe- flow in man and in guinea pigs. In a recently
cially the choroid), but as in other animals, no described animal model of lens-induced
degranulation was observed. An occasional uveitis in the rat,19 probably mediated by
leukocyte was present in the uveal tract, but immune complexes, eosinophils are also a
in no control eyes were cell observed in large prominent feature.
numbers; therefore no differential cell counts Our finding of large numbers of eosin-
were performed on controls. The most fre- ophils in the uveal tract in a systemic form of
quently observed infiltrating cell in control delayed hypersensitivity is not surprising
eyes was the lymphocyte, but an occasional considering our earlier observations of locally
plasma cell or neutrophil could also be found. induced delayed reactions in the guinea pig
Eosinophils were also observed occasionally, cornea and uveal tract.4'5 Although milder in
and almost every section had one or two intensity, the microscopic picture of the sys-
eosinophils in the choroid. temic reaction is qualitatively similar to the
locally induced reactions at the limbus and in
Discussion the uvea. The absence of basophils contrasts
Delayed hypersensitivity, or cell-mediated with the microscopic picture seen in the skin
immunity, is the major immunologic mech- and the central cornea in CBH reactions. We
anism in defense against many types of infec- previously suggested that certain local tissue
tion, in contact allergy, and in the rejection of factors, perhaps related to the microvascu-
tissue grafts and tumors. Delayed hypersen- lature, could influence the composition of the
sitivity responses are presumed to be impor- inflammatory cell infiltrate.4' 5 A second rea-
tant in many diseases affecting the eye either son for the paucity of basophils in the uveal
through local tissue damage or as part of a tract could be the abundance of mast cells
broader systemic process. They may be im- throughout the uvea. Mast cells secrete
portant in the pathogenesis of sympathet- eosinophil chemotactic factor of anaphylaxis
ic ophthalmia,9 Vogt-Koyanagi-Harada syn- (ECF-A), a substance chemotactic for eosin-
drome,10 and Behcet's disease.ll Delayed hy- ophils which is preformed in mast cells and
persensitivity responses are known to be de- basophils.20 Mast cells and basophils are be-
pressed in atopic conditions,12 herpes zos- lieved to have similar functions, and there is
ter,13 leprosy,14 sarcoidosis,15 and certain a well-known inverse relation between baso-
rheumatic disorders.16 phil and mast cell frequency in various spe-
Although eosinophils are generally consid- cies.21 Perhaps the already present large
ered a hallmark of allergic reactions involving population of mast cells obviates the need for
humoral immunity, they have only recently an influx of basophils.
been associated with delayed hypersensi- The role of the eosinophil and the reason
tivity responses.4"6 They are especially prom- for its prevalence in ocular delayed hyper-
inent in delayed hypersensitivity reactions sensitivity is uncertain. Eosinophils phago-
involving the eye, although they may be seen cytose immune complexes22 and are thought
within internal organs in a systemic form of to cause tissue injury in certain diseases.23
delayed hypersensitivity, SCBH. In human Recent investigations have identified a num-
ocular disease, eosinophils have been associ- ber of enzymes within eosinophil gran-
ated with sympathetic ophthalmia,17 cicatri- ules which are capable of inactivating the
cial pemphigoid, and atopic and vernal con- products of mast cell/basophil secretion.24
junctivitis.18 In many cases of sympathetic For example, arylsulfatase, histaminase, and
ophthalmia, eosinophils are concentrated in phospholipase D, which are found in eosino-
the inner choroid just under the choriocapil- phils, can inhibit slow-reacting substance of
laris.17 In the guinea pig, however, eosino- anaphylaxis, histamine, and platelet-acti-

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Volume 18
Number 9 Ocular findings in SCBH 969

vating factor, respectively. 25 These findings, 9. Wong, V.G., Anderson, R., and O'Brien, P.J.:
coupled with the lag of eosinophil influx by Sympathetic ophthalmia and lymphocyte transfor-
mation, Am. J. Ophthalmol. 72:960, 1971.
several hours following initial mediator re- 10. Hammer, H.: Lymphocyte transformation test in
lease, suggest a role for eosinophils in mod- sympathetic ophthalmitis and Vogt-Koyanagi-Har-
ulating or limiting allergic inflammation. The ada syndrome, Br. J. Ophthalmol. 55:850, 1971.
tissue eosinophilia which accompanies de- 11. Lehner, T.: Stimulation of lymphocyte transforma-
layed hypersensitivity responses in the eye tion by tissue homogenate in recurrent oral ulcer-
ation, Immunologyl3:159, 1967.
and in other organs could be secondary to the 12. McGready, S.J., and Buckley, R.H.: Depression of
activation and secretion of mast cells in these cell-mediated immunity in atopic eczema, J. Allergy
tissues. Although the mechanism for mast Clin. Immunol. 56:393, 1975.
cell mediator release does not appear to be 13. Morrison, W.L.: Viral warts, herpes simplex and
the extrusion of granules (as in IgE-mediated herpes zoster in patients with secondary immune
deficiencies and neoplasms, Br. J. Dermatol. 92:
anaphylaxis), a model in which a small quan- 625, 1975.
tum of intact or dissolved granule material 14. Waldorf, D.S., Sheagren, J.N., Trautman, J.R., et
flows to the cell surface, fuses with the al.: Impaired delayed hypersensitivity in patients
plasma membrane, and is discharged into the with lepromatous leprosy, Lancet 2:773, 1966.
extracellular space has been proposed. 26 The 15. James, D.G., Neville, E., and Walker, A.: Immu-
nology of sarcoidosis, Am. J. Med. 59:388, 1975.
actual mechanism for eosinophil chemotaxis
16. Williams, R.C., Jr., Debord, J.R., Mellbye, O.J., et
in uveal hypersensitivity reactions is not al.: Studies of B and T lymphocytes in patients with
presently known and will require further in- connective tissue diseases, J. Clin. Invest. 58:690,
vestigation. 1976.
17. Marak, G.E., Jr., Font, R.L., and Zimmerman,
L.E.: Histologic variations related to race in sympa-
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