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Effect of complement and polymorphonuclear leukocyte depletion on experimental skin lesions resembling systemic lupus erythematosus.

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581
EFFECT OF COMPLEMENT AND
POLYMORPHONUCLEAR LEUKOCYTE
DEPLETION ON EXPERIMENTAL
SKIN LESIONS RESEMBLING
SYSTEMIC LUPUS ERYTHEMATOSUS
P. G. NATALI, M. MOTTOLESE, and M. R. NICOTRA
Immunopathologic cutaneous lesions resembling human systemic lupus erythematosus (SLE) can
be induced in mice sensitized to ultraviolet (UV)-irradiated DNA following whole body irradiation with
UV light. T h e lesions are characterized by the formation of immune complexes at different skin sites. The
role played by cellular and humoral mediators in the
pathogenesis of this experimental model was investigated. The results obtained suggest that inflammation
that follows UV radiation is the major factor responsible for this pathology. Accordingly mice that were
rendered neutrophil (PMN) deficient did not manifest
skin lesions, and depletion of C3 complement component left them unchanged. I n addition time course
studies showed that PMN depletion did not prevent a
delayed skin involvement. Thus multiple factors seem
to mediate the onset of the immunopathologic changes
previously described.
Although native DNA is not antigenic in experimental animals, different kinds of physically and
From Laboratorio Irnrnunologia Istituto Regina Elena,
Rome, Italy.
Supported in part by Laboratorio Tecnologia Biornedica,
CNR, Italy.
Pier Giorgio Natali, M.D.: Istituto Regina Elena; M.
Mottolese, M.D.: Istituto Regina Elena; M. R. Nicotra, M.D.:
Istituto Regina Elena.
Address reprint requests to Dr. Natali, Istituto Regina
Elena, l’iale Regina Elena, 291, Rome, Italy.
Submitted for publication September 24, 1974; accepted
February 14, 1975.
Arthritis and Rheumatism, Vol. IS, No. 6 (November-December 1975)
chemically altered DNA molecules are able to induce
a specific immune response (1,2).
Native DNA irradiated with ultraviolet (UV)
light (UV-DNA) is highly immunogenic (3) and has
been used in experimental animal models to elucidate
some of the immunologic events leading to kidney (4)
and skin (5) involvement in systemic lupus erythematows in humans. Cutaneous lesions in particular could
be induced with high frequency in mice with circulating
antibodies to UV-DNA following exposure to UV light.
This treatment has been shown to be capable of inducing formation of the UV-DNA photoproduct,
thymidine dimer (6), and of releasing it from the skin
into the blood stream (7).
The skin immunopathology closely resembled
human lesions in that in vivo fixation of mouse 7globulins and C3 complement component to epidermal
cell nuclei and dermal-epidermal junction could be
observed together with accumulation of inflammatory
cells. In order to investigate the role played by cellular
and humoral mediators in the formation of these lesions, the effect of polymorpholeukocyte (PMN) and
complement (C3) depletion on the appearance of skin
immunopathology was investigated. Results of experiments reported here showed that PMN accumulation
in the skin, which follows UV light irradiation, is indispensable for the onset of the cutaneous pathology.
C3 depletion prevented only in vivo binding of the
complement component to the skin sites where mouse
y-globulins were bound to UV-DNA antigen.
582
NATAL1 E T AL
1
I
I
2
Days
3
4
Fig 1. Effect of nitrogen mustard injection (400 PglIOO g body
weight) on PMN number in C57/B1 (0-0) and Balb/C
(@-a) mice. PMN numberlmm’ is represented on the ordinate. Each point represents the mean of 5 mice per group.
Maximal depletion was reached 7 2 hours after drug injection.
A delayed binding of mouse 7-globulins t o epidermal cell nuclei was observed 24 hours after irradiation even i n the absence of circulating PAIN.
MATERIALS AND METHODS
Antigen Preparation and Animal Immunization. Native calf thymus DNA in highly polymerized form (Worthington Biochemical Corporation, Freehold, New Jersey) was
irradiated at a concentration of 500 &ml in phosphate
(0.01 M ) buffered saline (0.15 M ) with G30T8 germicidal
lamps (General Electric, Schenectady, New York) as previously reported (6).
After the irradiated DNA was complexed with an
equal amount of methylated bovine serum albumin
(MBSA), the complex was emulsified in Freund’s adjuvant
and injected subcutaneously once a week for at least 4 weeks
in either C57/B1 or Balb/C mice strains obtained from a local breeder. Because no difference in response to UV light had
been observed in previous studies between the two strains,
the experiments reported here were performed on either
black (C57/B1) or white (Balb/C) mice. After immunization
animals were Med and sera were tested for antibodies to
UV-DNA by indirect immunofluorescence as already reported (8.) Animals with antibody titers lower than 1/32
were not used.
Polymorph and C3 Depletion. Polymorph (PMN)
was depleted by injecting nitrogen mustard, methyl-bis-(Bchloroethyl) amine HCl, “Cloramin” (Simes, Milan, Italy),
in two separate intraperitoneal administrations 3 hours
apart for a total of 400 &lo0 g of body weight. T h e number of circulating PMN was monitored at different times
after red blood cell lysis with Turk-Losung liquid (Merck,
Darmstadt, West Germany), and by differential count on
blood smears stained with May-Grunwald reagent. Maximal
depletion of PMN, 50 to 100 cells/mmY, was reached in both
strains of mice at 72 hours and lasted at least 48 hours.
C3 was depleted in vivo by injecting purified cobra
venom factor (Cof) supplied by Cordis Laboratories, Miami,
Florida. The protein was injected intraperitoneally (20 U/
100 g of body weight). After a single injection of Cof, the
concentration of circulating C3 was estimated by quantitative immunodiffusion using 1 agarose (Seakem, Marine
Colloids, Rockland, Maine), 0.05 M EDTA plates, containing an appropriate concentration of a rabbit antimouse C3
specific antiserum (see below). Plates were left to develop at
room temperature for 18 hours in a moist chamber. The
radial immunodiffusion test was able to detect less than 10%
of normal serum C3 concentration. Loss of 90 to 95% of C3
was observed in both strains of mice 24 hours after Cof injection. Low levels of circulating C3 persisted for 2 days,
after which a gradual increase of this protein to normal
values was noticed.
Animal Irradiation. Mice were placed in individual
cages as previously described (5). The UV light sources were
two G30T8 germicidal lamps.
The distance from the light was 50 cm and the irradiation was continued for 7 hours. Before irradiation the
horny layer of the epidermis of the ears was stripped off
with adhesive tape in order to induce maximal UV-DNA
formation i n the epidermal and dermal nuclei. Because in
previous experiments most of the pathologic changes following UV light irradiation of the UV-DNA immunized
mice were detected in the skin of the ears, all the results
reported here were obtained from studies of this skin.
Tissue Sampling. Biopsies of the ears were taken
either immediately after 7 hours of UV light irradiation or
after 24 hours, under ether general anesthesia. All specimens were divided into two parts and processed for routine
histology (Bouin’s fixative) or snap frozen at -60°C in
Tissue-Tek O.C.T. compound (Ames Company, Elkhart,
Indiana) in a mixture of dry ice-acetone. Cryostat sections,
4 p thick, were used in direct and indirect immunofluorescence after being washed for 5 minutes in cold phosphatebuffered saline (PBS). A minimum of three nonconsecutive
sections were examined from each biopsy.
Reagent Antisera. Antisera to mouse and rabbit 7 S
immunoglobulins labeled with fluorescein isothiocyanate
were purchased from Hyland Laboratories, Los Angeles,
California. The sera appeared monospecific in immunoelectrophoretic analysis and at the dilution used showed an
F / P of 4 and a protein concentration of 1.7 mg/ml (antimouse-7-globulin antiserum) and F/P 2.5 with a protein
content of 0.8 mg/ml (antirabbit-7-globulin antiserum).
Rabbit antiserum to mouse C3 fraction of complement was
labeled with fluorescein isothiocyanate by the method of
Wood (10). A t the dilution used the serum had a F/P of 4
and a protein concentration of 0.7 mg/ml. Rabbit antiserum
to UV-DNA was obtained as previously described (6).
RESULTS
Effect of PMN and C3 Depletion on Histologic
Changes Induced by U V Light. Figure 1 shows the
effect of injection of nitrogen mustard on the number
of circulating polymorphs. W h e n 400 pg/IOO g of
body weight were administered i n two separate doses,
COMPLEMENT AND PMN LEUKOCYTE DEPLETION
0
t Cof
583
1
2
3
4
5
6
days
Fig 3. Serum C3 defiletion after injection of Cof (20 p l I 0 0 g
body weight) in C 5 7 / B l (0-0)
and BalblC (A-A)
mice.
Each point represents the mean of 4 mice per group. Low levels
of circulating C3 lasted 48 hours (black horizontal bar).
Fig 2. a ) Histology of the skin of the ears of PMN-deficient mice
after UV radiation. No inflammatory cells are detectable. H+E
stain (X 250). b ) Histologic feature of the skin of the ears of
C3 depleted mice after UV exposure. Large numbers of PMN
infiltrate the upper dermis. Vacuoliration and pyknosis of efiidermal cell nuclei are apparent. H+E stain (X 400).
a sharp decrease in the concentration of PMN was
observed both in C57/B1 and Balb/C mice. At 72
hours following drug injection an average of 50 to
100 PMN/mm3 could be detected. T h e low number
of neutrophils lasted for at least 48 hours.
When normal or UV-DNA immunized animals,
which were PMN-depleted, were irradiated according
to the experimental conditions that are known to induce a strong inflammatory response, the histologic
observation (Figure 2a) of the skin biopsies taken im-
mediately after irradiation showed no PMN accumulation either in the dermis or the epidermis. Few
scattered mononuclear cells could be detected in the
dermis. T h e epidermal cells showed only minor
changes consisting mainly of pyknosis and vacuolization on the side of maximal exposure. These findings
contrasted sharply with the effects of in vivo depletion
of C3 complement component. Mice injected with Cof
showed after 24 hours a reduction of C3 to a concentration less than 10% of normal (Figure 3). When
C3-depleted mice were exposed to UV light a normal
inflammatory response in the skin was observable.
Histologic studies (Figure 2b) showed neutrophils accumulated in large numbers all through the dermis,
filling dermal vessels and in close contact with the
dermal-epidermal junction. In this instance epidermal
cells showed scattered pyknotic nuclei in the basal
layer.
Incidence and Patterns of in vivo Bound Ig and
Complement in the Skin of Nitrogen Mustard- and
Cof-Treated Mice. Table 1 summarizes the patterns
and incidence of skin fixation of mouse Ig and C3.
UV-DNA immunized mice that were UV light irradiated showed maximal incidence of immunopathologic
lesions in the skin of the ears immediately after exposure. Direct immunofluorescence studies showed
mouse Ig (lOO~o)and C3 (60y0)bound in vivo to epidermal and dermal cell nuclei (Figure 4a). Fixation of
the same immune reactants at the dermal-epidermal
NATAL1 E T AL
Table 1. Eflect of PMN and C3 Depletion on Zn Vivo Bound
Mouse y-Globulins and C3 in Zmmune Mice
Irradiated w i t h UV Light
Patterns of Skin Fixation*
Dermal Speckled
and Reticular
Untreated
Mouse y-globulin
Mouse C3
UV-DNA
PMN depleted
Mouse 7-globulin
Mouse C 3
UV-DNA
C3 depleted
Mouse y-globulin
Mouse C3
UV-DNA
Nuclear
5 / 5 (100)
3 / 5 (60)
515 (100)
015 (0)
015 (0)
5 / 5 (100)
5 / 5 (100)
0 / 5 (0)
515 (100)
*Ear biopsies taken immediately after UV exposure.
+Number of positive per number tested (yopositive).
junction (Figure 4b) occurred in 60y0 of the biopsies
for mouse Ig and in 40% of those for mouse C3. As
expected the antigen UV-DNA was present in all skin
biopsies (Figure 4c). Quite striking differences were
observed with immune animals that were PMN deficient. UV radiation in these mice, although able
to induce the formation of UV-DNA antigen in
skin nuclei, did not show in vivo fixation of mouse
Ig and C3 either on nuclei or at the dermal-epidermal
junction.
On the other hand, mice that received Cof and
at the time of UV light irradiation had only 10% of
the original C3 concentration did not manifest any in
vivo binding of the complement component at tissue
sites. C3 depletion did not interfere with the amount
of mouse anti-UV-DNA antibody that reacted with the
homologous antigen, which was induced in all the
animals. Nuclear fixation of immunoglobulins was in
fact present in 1 0 0 ~ of
o the C3-deficient mice.
Time Course Study of Skin Lesions in Polymorph-Deficient Mice. I n order to determine whether
suppression of immunopathologic lesions in mice that
were PMN depleted persisted over a period of time,
skin biopsies were sampled in a group of mice both
at the end of irradiation and 24 hours later. T h e results of this experiment are summarized in Table 2.
Although skin biopsies collected immediately
after UV light radiation did not manifest any in vivo
binding of either immunoglobulins or C3 (Figure 5a),
tissue samples taken in adjacent sites after 24 hours
demonstrated in all cases mouse 7-globulins fixed to
epidermal cell nuclei (Figure 5b). Dermal-epidermal
Fig 4. a ) Direct iinniiinofliiorescence showing in vivo binding of
mouse y-globulins at the epidermal nuclei in UV-immunized,
UV light-irradiated mice ( X 250). b ) Reticular distribution of
y-globulins in the upper dermis and at the dermal-epidermal
junction in the same animals ( X 250). c ) Zndirect immunopuorescence to detect U V - D N A in the epidermis of UV light-irradiated
mice. Epidermal and dermal nuclei show bright fluorescence
after reaction w i t h a rabbit anti-UV-DNA antiserum (X 250).
junction stain was very seldomly observed. T h e extent
of the lesions was generally limited to some areas of
the epidermis with no appreciable involvement of the
deep dermal structures. No C3 was detectable with
the same distribution. These findings strongly suggested that, although neutrophils were essential to the
onset of the early lesion, they were completely unrelated to the appearance of the immunopathology
observed 24 hours after irradiation. In fact the number of peripheral neutrophils in most cases was further
decreased or unchanged. Titers of circulating antibodies to UV-DNA also seemed not to play any relevant role in such phenomena.
DISCUSSION
Formation of immune complexes either in circulation or at tissue sites appears to be one of the most
COMPLEMENT AND PMN LEUKOCYTE DEPLETION
585
of inflammatory cells together with in vivo deposition
of mouse y-globulins and C3 to different skin sites.
Because complement activation and neutrophil accumulation are two important phenomena in immune
inflammation, the effect of their suppression in vivo in
this animal model was investigated. Depletion of the
third complement component achieved by Cof did not
interfere with the binding of circulating mouse antiUV-DNA antibodies to the homologous antigen in the
skin or with accumulation of neutrophils. This finding
suggests that complement activation plays only a secondary role in eliciting these skin lesions. UV light
has the ability to induce increased vascular permeability and PMN accumulation independently from
the presence of a specific immune reaction at the skin
level (5).
Mice that were PMN deficient, on the contrary,
completely lacked any immunopathologic manifestation. No in vivo fixation of mouse 7-globulins to the
skin was observable after UV light exposure. This result can also be explained by the fact that UV light
is a strong inflammatory stimulus mainly mediated by
polymorpholeukocytes (12).
T h e accumulation of these cells together with
the increase in vascular permeability may be induced
by UV light through various nonimmune mechanisms
involving changes of cell membrane permeability (13),
liberation of vasoactive amines from tissue reservoirs
(14) and disruption of lysosomes of pre-existing and
accumulating cells (15,16) with liberation of enzymatic
activities and leukotactic factors (17).
Although nitrogen mustard does not selectively
affect leukocyte subclasses, the clrug does subtract the
cell population that is primarily engaged in the UV
light inflammatory response by depleting most of the
circulating polymorphs. Such response is likely to allow circulating anti-UV-DNA antibodies to react with
newly formed UV-DNA antigen, adding a further in-
Fig 5. a ) Lack of any in vivo binding of mouse y-globulins in
biopsies collected immediately after U V radiation in PMN-deficient mice (X 250). Autofluorescence of the upper epidermis is
present. b ) Ear biopsies taken from adjacent sites 24 hours after
UV light show mouse y-globulins fixed to epidermal nuclei
( X 250).
common mechanisms that bring about various cellular
alterations such as necrosis, proliferation, inflammatory cell accumulation, and increased vascular permeability (1 1). T h e skin lesions previously described
(5) were shown to be mediated presumably by the
formation of immune complexes between UV-DNA
antigen and homologous antibody. Thus the simultaneous presence of both immune reagents was indispensable for the appearance of cutaneous pathology.
This appearance was characterized by a n accumulation
Table 2. Time-Course of Skin Lesions in PMN-Depleted Mice after UV Exposure
0 Hours
24 Hours
Animal
No.
y-Globulin
c3
1
2
-
-
32
50
3
4
-
-
64
64
64
150
200
200
64
50
5
-
Ab*
UV-DNA
-
*Reciprocal of serum antibody titerto UV-DNA.
iIn vivo binding to epidermal cell nuclei.
PMN/mms
y-Globulin+
+
+
+
+
+
c3
-
Ab*
UV-DNA
-
32
64
-
64
-
PMN/mms
60
100
150
64
50
32
50
586
NATAL1 E T AL
flammatory insult of immune nature. I t should be
stressed that the nonselective activity of the drug does
not exclude a possible participation of other humoral
and/or cell-mediated mechanisms in the onset of this
pathology, although at the high levels of exciting radiation delivered to the animals polymorph accumulation
should be of pathogenetic importance.
Despite the low number of circulating PMN,
binding of anti-UV-DNA antibodies to cell nuclei was
again observed in the nitrogen mustard-treated mice
24 hours after irradiation. This finding agrees with
previous studies that demonstrated that the increase
in vascular permeability following moderate doses of
UV light radiation is characterized by a diphasic response (17). T h e first response is mediated through
liberation of amines such as histamine (guinea pig)
and 5-hydroxytryptamine (rats) and is accompanied by
emigration of leukocytes; the second occurs 24 hours
after the UV stimulus and is completely independent
of the presence of inflammatory cells. T h e mediators
of this delayed permeability increase remain unknown.
T h e failure of C3 to bind at 24 hours to the
epidermal cell nuclei is not yet understood. Although
nitrogen mustard did not reduce C3 serum levels, an
effect of the drug on other serum components that
participate in C3 activation cannot be excluded. T h e
data presented here suggest that this experimental
model may provide a tool for gaining more insight
into the mechanism involved in the autoimmune manifestations of SLE. T h e same model may also help to
explain the role that different humoral and cellular
mediators have in this cutaneous pathology. Such
knowledge will be valuable in guiding the therapeutic
approach to this disease.
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Natl Acad Sci USA 52:279-285, 1964
2. Van Vunakis H, Seaman E, Kahan L, et al: Formation
of an adduct with tris (hydroxymethyl) amino ethane
during the photoxidation of deoxyribonucleic and
guanidine derivatives. Biochemistry 5:3986-3991, 1966
3. Natali PG, T a n EM: Antibodies to photoproducts of
DNA irradiated with ultraviolet light. Fed Proc 28:696,
1969
4. Natali PG, T a n EM: Experimental renal disease induced by DNA anti-DNA immune complexes. J Clin
Invest 51:345-355, 1972
5. Natali PG, T a n EM: Experimental skin lesions in mice
resembling systemic lupus erythematosus. Arthritis
Rheum 16:579-589, 1973
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46:506-5 18, 1971
7. T a n EM: Production of potentially antigenic DNA in
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10. Wood BT, Thompson SH, Goldstein G : Fluorescent
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11. Dixon FJ: T h e role of antigen-antibody complexes in
disease. Harvey Lect 58:21-52, 1962-1963
12. Logan G, Wilhelm DL: T h e inflammatory reaction in
ultraviolet injury. Br J Exp Pathol 46:286-299, 1966
13. Kinaldi RA: T h e effect of ultraviolet radiation on the
hydration of amoeba proteus as determined by contractile vacuolar function. Exp Cell Res 18:62-69, 1959
14. Logan G, Wilhelm DL: Vascular permeability changes
in inflammation. I. T h e role of endogeneous permeability factors in ultraviolet injury. Br J Exp Pathol 47:300314, 1966
15. Johnson BE: Ultraviolet radiation and lysosomes in
skin. Nature 219: 1258-1259, 1968
16. Desai ID, Savant PL, Tappel AL: Peroxidative and radiation damage to isolated lysosomes. Biochem Biophys
Acta 86:277-285, 1964
17. Janoff A, Zweifach BW: Production of inflammatory
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