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Reticuloendothelial phagocytic function and antibody formation in gunn rats
Volume
EXPERIMENT.\L
AND
MOLECULAR
PATHOLOGY
Reticuloendothelial
Phagocytic Formation
B. B. LOZZIO, E. A. The
University
Tennessee and
Tennessee
Nacionales
of
Medicine,
Department
Loukiana
70116
(1971)
Function
in Gunn MACHADO;
Memorial
Institutos
School
Orleans,
of 37920,
14, 57-G6
Research de la Salud
Received
AND Center (E.A.M.),
of Physiology
August
14, No. 1, February, 1971 Printed in U.S.A.
and
Antibody
Rats’ R. DI LUZIO
N. and
Hospital Haedo,
(N.R.D.),
(B.B.L.), Buenos
Tulane
Aires,
Knoxville,
Argentina,
University,
New
%?,I970
The reticuloendothelial system (RES) function was determined in Gunn rats by the carbon-gel clearance and the RE 1311 lipid emulsion test. Microscopic observations and antigenic stimulation with sheep erythrocytes were also performed. Antibody formation was followed at the cellular and humoral levels by the hemolytic plaque technique and by agglutinin and hemolysin titrations, respectively. Gunn rats have hepatomegaly compared to the parent strain, the Wistar rats. Splenomegaly was only observed in homozygous females. Stimulation of the RES activity. as denoted by increased vascular clearance and hepatic and splenic upt.ake of the lipid emulsion, was observed in female Gunn rats. Only homozygous females manifested RES stimulation as determined by the carbon method. The R.ES carbon phagocytic function was normal 7 and 30 days after splenectomy or ovariectomy, respectively. In Gunn rats, the Kupffer cells and splenic follicles appeared hypertrophic. The marginal zone was enlarged and hyperplasia of REcells was present in the red pulp. Although similar humoral antibody titers were found, the number of splenic antibody forming cells was greater in Wistar than in Gunn rats. The RES hyperfunction of female Gunn rats seems to be associated with hypersplenic function and probably with a disturbance of estrogen metabolism. The hypertrophy of the lymphoid follicles and hyperplasia of reticulum cells and macrophages in the spleen of Gunn rats did not provide more cells able to respond to antigenic stimulation. In this regard, either fewer antibody-forming cells were sufficient to produce amounts of agglutinin and hemolysin similar to those of Wistar rats. or other areas of the lymphopoietic system compensated for the diminished numher of antibody-forming cells in the spleen of Gunn rats.
The Gunn strain of rats (Gunn, 1938) is a mutation of the Wistar rat characterized by the presence of jaundice secondary to the absence of liver bilirubin glucuronyl t’ransferase activity. Although Gunn rats have been used extensively as an experimental model for studies of bilirubin metabolism, functions other than those related to the defect of bilirubin conjugation have not yet been investigated (reviewed by Housset et al., 1967). It has recently been found that jaundiced females of our Gunn rat colony have splenomegaly and anemia caused by enhanced splenic erythrophagocytosis (Loz’ Supported in part by U.S.P.H.S. 2 Career award from the Division Cientificas y Tdcnicas, Buenos Aires,
Grant AM-12490. of Medical Science. Argentina. 57
Consejo
National
de Investigacioncs
58 zio et al., gous and hyperplasia structure formation
LOZZIO,
MACHADO,
AND
DI
LUZIO
1970). Preliminary microscopic examination of the spleens of homozyheterozygous rats revealed hypertrophy of the eplenic follicles and of the red pulp. These observations led to an investigation of the and function of the reticuloendothclial system (RES) and antibody in Gunn rats. IIATERIAL
ASD
METHODS
Wistar inbred strain of rats (Microbiological Assoc., Walkersville, Naryland) as well as heterozygous (jJ) and homozygous (jj) Gunn rats from the Cniveraity of Tennessee 3Lemorial Research Center colony (Lozzio et al., 1967) were used in these studies. Both strains of rats were free of Barto?zelln UZW~S. Sixteen milligrams of oxytetracyclinc hydrochloride (Terramycin, Animal Formula, Charles Pfizer, New York, S.Y.) per 100 ml of chlorinated drinking water were used prophylactically to prevent chronic pneumonia (PPLO) in the Wistar and Gunn rats. Male and female rats, 21/z to 4 months old (170-300 gm body weight), were fed a standard diet (Purina Lab Chow). Gunn rats found to hare renal lesions on aut.opsy were excluded (Lozzio et al., 1967). Evaluation of the RES function by the colloidal carbon technique was performed under sodium pentobarbital anesthesia (4 mg/lOO gnlj. Ether anesthesia was employed when phagocytic function was evaluated by using the gelat,inized “RE test lipid emulsion.” Phagocytic function was measured by determining the intravascular clearance rate of gelatinized 1311-triolein labeled “RE test lipid emulsion” as previously described (Di Luzio and Riggi, 1964). The “RE test lipid emulsion” was injected intravenously at a triglyceride dose of 25 mg/lOO gm body weight. Following the injection, five serial O.l-ml aliquots of whole blood were obtained from the tail at approximately 2-minute intervals and evaluated for total radioactivity. Blood radioactivity per milliliter blood, expressed as the percentage of t,he injected dose was plotted semilogarithmically against time in minutes ancl half-times t’tllS were determined. Liver, lung, and spleen tissue distribution of the labeled emulsion was determined on aliquots of organs taken 10 minutes after injection of the test colloid. Tissue uptake is expressed as the percentage of the injected dose per total organ (%ID/TO) and as the percentage of the injected close per gram tissue (%rro/gm) iDi Luzio and Riggi, 1964). The RES phagocytic function was also determined by the carbon-gel clearance technique as described elsewhere (T,ozzio, 1967a, 1967b). Animals w-cre injected intravenously with 8 me;/100 gm of body weight’ of colloidal carbon. Blood samples (50 ~1 each) were taken every 3 minutes for 15 minutes. Carbon concentrations were determined spectrophotometrically in the hemolyzed blood, plotted semilogarithmically against, time in minute,, q and half-times were calculated. The primary immune response to sheep erythrocytes was studied by injecting 1 ml of a 20% suspension of sheep erythrocytes intravenously. Animals were bled daily from days 5 to 7 and at 2 or 3-day intervals for 15 days after immunization. Serum samples were stored at -20°C until titrated. Serum complement was inactivated at 56°C for 30 minutes. Hemagglutinin and hemolysin titers were
PHAGOCYTOSIS
AKD
IMMUNE
RESPONSE
IN
GUNN
RATS
59
determined by serial twofold dilutions of inactivated serum as described elsewhere (Lozzio and Comas, 1969). Titers were expressed as the loga of the reciprocal of the highest dilution giving visible agglutination or complete hemolysis, respect.ively. The pattern of immunologic response was also evaluated by the plaque assay technique of Jerne et al. (1963). Rats were immunized as indicated previously and were sacrificed by total exsanguination at days 3, 6, and 12 after antigenic stimulation. The spleen was removed and weighed. Two pieces, weighing approximately 100 mg each, were gently teased and t.he cells suspended in 2 ml of Eagle’s medium. Nucleated cell counts were performed at this stage on each cell suspension. Since it was found that the number of cells in a piece of spleen tissue is proportional to its weight, the tot.al number of spleen cells was calculated on the basis of the average cell number found in two pieces of splenic tissue, multiplied by the total weight of the organ. This method has an error of no greater than lo%, and obviates the cumbersome and time consuming teasing of the whole spleen of the rat. The cell suspensions were diluted 1: 100 and 1:250. The number of spleen cells producing specific antibodies against sheep erythrocytes was determined by direct (19s) hemolytic plaque formation in agar gel, using duplicate aliquots (0.5 ml) of each dilution. The average of four determinations was expressed as plaque counts per million spleen cells, or per whole spleen. Microscopic examination was performed on specimens of the liver, spleen, lungs, bone marrow, lymph nodes, kidneys, and adrenal glands. Tissues were fixed in 10% phosphate buffered formaldehyde (pH 7.0) solution and processed by standard techniques. Sections were stained with hematoxylin-eosin and PAS. Iron was analyzed as indicated by Gomori (1953). Serum bilirubin concent.rations were determined by the diazo reaction. RESULTS The liver and spleen weights were similar in Wistar rats of both sexes (Table I). A significant (p < .OOl) enlargement of the liver was observed in jJ and jj Gunn rats of both sexes when compared with Wistar rats. Ninety per cent of the jj females also had significant (p < .OOl) splenomegaly when compared with other groups of rats studied. The kidneys appeared significantly (p < .Ol) enlarged in jj rats of both sexes. There was no difference in the weight of the lungs. Ovariectomy or splenectomy did not influence the weight of the liver in jJ and jj female Gunn rats. Splenomegaly was unchanged in ovariectomized jj animals. Bilirubin was not detectable in the serum of Wistar and jJ Gunn rats. A mean serum bilirubin concentration of 6.45 f 0.36 mg % was found in 80 jj animals of both sexes with a range of 5.2 to 9.0 mg %. The intravascular clearances (h/z) of the triolein 1311-labeled emulsion were nearly identical (13.1-14.6 minutes) in male and female Wistar rats (Table II). The removal of lipid emulsion was markedly enhanced in jj (5.7 minutes) and jJ (9.2 minutes) females of the Gunn strain when compared with either their male
60
LOZZIO.
M3CHdDO.
3KD
TABLE ORGAS
WEIGHTS
ANL)
Homozygous Male Female
Liver
10 10
3.32 3.GG
f f
25 24
4.09 4.58
2G 40
4.18 4.87
weight
Spleen
0.09a 0.08
Kidneys
Lungs
0.20 0.25
f f
0.01 0.01
0.M f O.BO f
0.02 0.03
0.74 0.73
* *
0.02 0.02
f 0.3-l III 0.30
0.24 0.26
* f
0.07 0.01
0.51. f O.GO i
0.008 0.01
0.83 0.78
zt 0.01 32 0.03
f f
0.25 0.37
z!z 0.01 + 0.01
0.61 O.F&
0.02 0.02
0.89 0.92
zt 0.02 f 0.03
Gmm
aMeanf
0.12 0.06
* f
1SE TllBLE CLEARAXE
AND
TISSUE
EMULSION”
II
I~ISTRIBUTION IN
WISTAR
OF AND
THE
No. of rats
Group
Liver 74, m/TO
Wistar Male
TEST
LIPID
R.\Ts uptake
r% ID)
Lungs
I % m/m
“RF:
GE.,.ITIKIZED
GUNN Organ
Female Heterozygous Male Female Homorygous Male FlXTl&
1 ATS
Gunn
Female
VASCULAR
GUKN
70 of body
of rats
Wist,ar Male Female Heteroxygous Male
LUZIO
I
OF WISTAR
I 0. Y
Group
DI
Spleen
70ID/TO
__w ID/‘:*
14.6 f l.4a 13.1 * 2.2
36.8 It 3.G 5.0 f 47.6 f 2.7 4.9 i
0.7 0.1
1.4 f 0.1 1.1 + 0.6
1.1 z!z 0.2 1.0 rt 0.1
3.9 * 0.4 8.3 + 0.6
7.3 l 15.3 f
16.4 f 9.5 *
47.5 zk 3.7; 4.5 f 50.3 * 4.1 7.0 f
0.4 0.5
1.5 f 1.7 f
1.1 i 1.3 f
4.0 * 0.3 4.8 * 0.2
6.5 zt 2.2 10.5 f 0.6
40.5 f 60.5 f
0.6 1.0
1.4 zk 0.1 1.5 i 0.1
0.6
2.7
Gunn 5.7 0.9
0.1 0.1
0.1 0.1
Gunn 14.4 zt 1.8 5.7 f 1.8
3.1 5.0
3.5 f 7.3 i
1.1 * 0.1 1.4 f 0.2
4.4 * 3.9 f
0.2 0.4
i.4 x.i
rt 0.3 i 1.7
counterparts, or wit’h Wistar rats. The increase in vascular clearance in female Gunn rats appears related, in part, to increased liver uptake as denoted by the %In/gm values. The uptakes of the lungs and spleen were unaltered in Gunn rats as determined on either a total organ basis or on the basis of %ID/gDl. The rate of carbon-gel clearance (Table III) was equivalent in Wistar rats, jJ animals of both sexes and jj male Gunn rats. In contrast, a mild but constant and significant (p < .OOl) acceleration of the rate of carbon clearance was observed in the majority of Jj female Gunn rats. The RES carbon phagocytic function was normal shortly (7 days) aft’cr splenectomy, while a 30-day period was necessary to achieve the normalizat’ion of phagocytosis in ovariectomixed jj animals. The same surgical procedure P did not alter phagocytoeis of colloidal carbon in i-J female animals. The appearance of the reticuloendothclial cells (RE) in different. organs of the Gunn rat resembles that of the RES under moderate stimulation. The Kupffer
PHAGOCYTOSIS
SNI)
IMMUNE
RESPONSE
TABLE IUS
C.\KHOK
PH.\GOCYTIC c;UNN
GUNN
RATS
61
III ACTIVITY
OF WIGT.IE
.LND
R .\TS NO.
of rats Wistar Male Female Heteruygous Malt Female Homo:.ygous Male Female Splenectomized Heterozygow Homozypous Ovariecton~ized Heterozygous Heterozjgu,ls Homozygotw IIomozygou~
IN
10 10
Days after surgery
-
Carbon clearance (t112, minutes)
12.8 12.3
* +
0.2ou 0.26
20 20
11.0 12.4
+ +
0.3-l 0.55
20 20
12.9 8.6
+ *
0.50 0.31
13.4 12.3
Zt 0.19 k 0.09
12.5 l-k.1 10.0 15.2
+ * f f
Gunn
GUMI
Female 10
i
10
7
10
7
10 10 10
30 i 30
0.10 0.16 0.21 0.16
cells appeared hyl)ertrophic (Fig. 2). The majority had a round or oval shape instead of the typical triangular or stellate shape characteristic of those cells in Wistar rats (Fig. 1) . The anatomical distribution of Kupffer cells was preserved. Phagocytoeis of carbon by Kupffer and other liver reticuloendothelial cells was proportional to their size and number. A markedly increased number of reticulum cells and macrophages was observed in the spleen of Gunn rats of both sexes as compared with the spleens of JYistar rats (Fig. 3). The picture in the red pulp was characterized by the presence of an abnormal number of active macrophages containing larger iron &ores than those of Wistar rats. In Gunn rats, hypertrophy of the endothelial sinus lining cells caused narrowing of the sinusoidal lumen. Numerous reticulum cells populated the hypertrophic splenic follicles (Fig. 4). The marginal zone was enlarged by the presence of an abnormal number of macrophages and reticulum cells. A marked increase of splenic carbon granulopexy was found when compared with JVirtar rats. A similar picture, characterized by an increased number of cells able to phagocytize carbon, was observed in the lungs, bone marrow, lymph nodes, adrenal glands, and kidneys. Natural antibodies to sheep erythrocyten were not found in t’he serum of the rats studied. The agglutinin and hemolysin levels in Wistar and Gunn rats attained a maximum titer 4-7 clays after antigenic stimulation, and no significant differences of the humoral antibody titers were observed between the two strains (Fig. 5 ) . As shown in Table IV, the peak of plaque-forming cells (PFC) occurred on
62
LOZZIO,
MACHADO.
AND
DI
LUZIO
The lxeswt~ stutlic>s in~lic:~tt~ t,hat ( lure r:its 11:/w hqmtonwgaly. :m alteration of the number ant1 :wtivit\of RES wlls, :mtl a tlin~inishetl numlwr of splenic antibody-forming ~11s n-htw coinpared with their lmrcwt strain, the Wistar rat. In addition, ij ft9wlcs niaiiifcstetl s.plent~niegwlpand both j.J ant1 jj femalw sl~owctl stimulation of RIM phagocytic actkit?- :w clc~tcrmiutd eithcxr I)\- “RN lipid emulsion” or carbon-gel methods.
PHAGOCYTOSIS
AND
IMMUNE
RESPONSE
IN
GUNN
FIG,. 3. Srction of the spleen from a female Wistnr rat marginal zone (MZ) and red pulp. (RP). H.E. X 200. FIN. 4. Section of the spleen from a homozygous frmnle t!!c splcnic follicles (SF) and enlargrmrnt of thrl mnrginal wt=n (RP). H.E. X 200.
?howin
g the
Gwm xon?
RATS
splenir
ant. s!:owing (MZ). The
63
follicles
(SF).
l~ypertrol~lly of wtl pulp is also
As a consecpence of the bilirubin transferase deficiency, jj Gunn rats have permanently high blood and tissue levels of bilirubin which is bound to albumin and other protein fractions (Housset et al., 1967 1. Since albumin-bound bilirubin exerts no harmful effects on mammalian cells iCowgcr et nl., 1965; Kikuchi, 1961) and some of the pathophysiologic changes described in this study were present in both j,J and jj animals, t’he increased amount’ of bilirubin was probably not the cause of the alterations noted. Bartonellosis can be ruled out as a cause of the splcnomegaly and the hematologic alterations previously described (Lozzio et nl., 1970) because the animals, on repeat,ed examination, were found to be frw of Rartonelln murk. Both Wistar
-
WISTAR
(26)
l
4
GUNN
(34)
HEMOLYSIN
234567 2
9
I
AGGLUTININ
(2
(5
PHAGOCYTOSIS
AND
IMMUNE
RESPONSE
IN
GUNN
RATS
65
The RES hyperfunction found in fcmalc Gum rats was associated with hepatomegaly. However, the hepatomcgaly doe.+ not seem to be the sole cause of the RES hyperfunction because jJ and jj malts had hepatomegaly and normal phagocytic function. Normal removal of carbon from the circulation was observed in Ii females 7 days after splenectomy or 30 days after ovariect’omy. Moreover, jj female Gunn rats have a syndrome of hypersplenism involving the erythrocytee. It is characterized by mild anemia and shortened erythrocyte survival time, caused by augmented splenic sequestration (Lozzio et nl., 1970). Therefore sex differences and splcnic hyperactivity remain as l)ossible factors in the pathogenesis of the anemia (Lozzio et nL.. 19701 and of the alteration of the RES phagocytic function. It has long been known that splcncctomy combined with ovariectomy creates the optimum conditions for the expcrimcntal blockade of the R,ES (Leites and Riabow, 1928). Recent studies inclicat,e that the eplee~~ is a significant RE organ 3s denoted by the decreawl clearance rate of the “RE lipid emulsion” in splenectomized rats (Patterson et nl., 1970). The mechanism by which the spleen influences RES phagocytic funct’ion is unknown. The effects of estrogens on the RES hare hecn recently reviewed (,VernonRoberts, 1969). RES act’ivity varies with the estrous cycle and falls after ovariectomy. Conversely, the administration of natural and synthetic estrogens produces hepatosplenomcgaly due to a striking hyperplasia of RE cells associated with hyperphagocytic activity (Vernon-Roberts, 1969). In t,hc present study, Gunn rat s were noted to have hypertrophy of liver macrophages and hel)atomegaly. Hyperplasia of splenic RE cells was found in all rats of the Gum strain, including those with normal RE phagocytic function. Studies demonstrating that splenectomy or ovariectomy in jj females suppress the hyperphagocytic function confirm previous observations on the role of the spleen in RES activity. At the same t)ime, they provide indirect evidence for the involvement of estrogen in RES activity even though ovariectomy did not affect splenomegaly in thwc rats. The fact that infertility occurs in all jj female Gunn rats further suggests a disturbance of sex hormone metabolism. Although splenic follicles alqwarcd hypertrophic and hypcrplasia of the reticulum cells and of the macrophages was present in Gunn rats, fewer antibody form’ng cells were found in the spleens of Gunn rats than in the spleens of Wirtar rats. This finding indicates that the proliferation of certain cell lines does not neces::arily make available a grcatcr number of cells able to react with the antigen. In fact, the hypcrtrophy of the lymphoirl follicles and the RE hyperplasia would sew1 to bc detrimental to iInmunocompt~t~~tlt cells since there were more splcnic cclla after antigenic stimulation in Gunn than in Wistar rats, while the number of plaque-forming cells was diminished. Since the splecm is the principal organ of hemolysin formation in the rat (Rowley, 1950) it appears that the number of splenic antibody-forming cells obs;cr\-ed in Gunn rats was sufficient rats to maint’ain similar agglutinin and hemolysin titers to those of Wistar throughout, the primary immune response. It is also probable that other areas of the lymphopoietic system compensate for the diminished number of antibodies formed in the spleen of Gunn rat’s.
EXPERIMENT.\L
AND
MOLECULAR
PATHOLOGY
Reticuloendothelial
Phagocytic Formation
B. B. LOZZIO, E. A. The
University
Tennessee and
Tennessee
Nacionales
of
Medicine,
Department
Loukiana
70116
(1971)
Function
in Gunn MACHADO;
Memorial
Institutos
School
Orleans,
of 37920,
14, 57-G6
Research de la Salud
Received
AND Center (E.A.M.),
of Physiology
August
14, No. 1, February, 1971 Printed in U.S.A.
and
Antibody
Rats’ R. DI LUZIO
N. and
Hospital Haedo,
(N.R.D.),
(B.B.L.), Buenos
Tulane
Aires,
Knoxville,
Argentina,
University,
New
%?,I970
The reticuloendothelial system (RES) function was determined in Gunn rats by the carbon-gel clearance and the RE 1311 lipid emulsion test. Microscopic observations and antigenic stimulation with sheep erythrocytes were also performed. Antibody formation was followed at the cellular and humoral levels by the hemolytic plaque technique and by agglutinin and hemolysin titrations, respectively. Gunn rats have hepatomegaly compared to the parent strain, the Wistar rats. Splenomegaly was only observed in homozygous females. Stimulation of the RES activity. as denoted by increased vascular clearance and hepatic and splenic upt.ake of the lipid emulsion, was observed in female Gunn rats. Only homozygous females manifested RES stimulation as determined by the carbon method. The R.ES carbon phagocytic function was normal 7 and 30 days after splenectomy or ovariectomy, respectively. In Gunn rats, the Kupffer cells and splenic follicles appeared hypertrophic. The marginal zone was enlarged and hyperplasia of REcells was present in the red pulp. Although similar humoral antibody titers were found, the number of splenic antibody forming cells was greater in Wistar than in Gunn rats. The RES hyperfunction of female Gunn rats seems to be associated with hypersplenic function and probably with a disturbance of estrogen metabolism. The hypertrophy of the lymphoid follicles and hyperplasia of reticulum cells and macrophages in the spleen of Gunn rats did not provide more cells able to respond to antigenic stimulation. In this regard, either fewer antibody-forming cells were sufficient to produce amounts of agglutinin and hemolysin similar to those of Wistar rats. or other areas of the lymphopoietic system compensated for the diminished numher of antibody-forming cells in the spleen of Gunn rats.
The Gunn strain of rats (Gunn, 1938) is a mutation of the Wistar rat characterized by the presence of jaundice secondary to the absence of liver bilirubin glucuronyl t’ransferase activity. Although Gunn rats have been used extensively as an experimental model for studies of bilirubin metabolism, functions other than those related to the defect of bilirubin conjugation have not yet been investigated (reviewed by Housset et al., 1967). It has recently been found that jaundiced females of our Gunn rat colony have splenomegaly and anemia caused by enhanced splenic erythrophagocytosis (Loz’ Supported in part by U.S.P.H.S. 2 Career award from the Division Cientificas y Tdcnicas, Buenos Aires,
Grant AM-12490. of Medical Science. Argentina. 57
Consejo
National
de Investigacioncs
58 zio et al., gous and hyperplasia structure formation
LOZZIO,
MACHADO,
AND
DI
LUZIO
1970). Preliminary microscopic examination of the spleens of homozyheterozygous rats revealed hypertrophy of the eplenic follicles and of the red pulp. These observations led to an investigation of the and function of the reticuloendothclial system (RES) and antibody in Gunn rats. IIATERIAL
ASD
METHODS
Wistar inbred strain of rats (Microbiological Assoc., Walkersville, Naryland) as well as heterozygous (jJ) and homozygous (jj) Gunn rats from the Cniveraity of Tennessee 3Lemorial Research Center colony (Lozzio et al., 1967) were used in these studies. Both strains of rats were free of Barto?zelln UZW~S. Sixteen milligrams of oxytetracyclinc hydrochloride (Terramycin, Animal Formula, Charles Pfizer, New York, S.Y.) per 100 ml of chlorinated drinking water were used prophylactically to prevent chronic pneumonia (PPLO) in the Wistar and Gunn rats. Male and female rats, 21/z to 4 months old (170-300 gm body weight), were fed a standard diet (Purina Lab Chow). Gunn rats found to hare renal lesions on aut.opsy were excluded (Lozzio et al., 1967). Evaluation of the RES function by the colloidal carbon technique was performed under sodium pentobarbital anesthesia (4 mg/lOO gnlj. Ether anesthesia was employed when phagocytic function was evaluated by using the gelat,inized “RE test lipid emulsion.” Phagocytic function was measured by determining the intravascular clearance rate of gelatinized 1311-triolein labeled “RE test lipid emulsion” as previously described (Di Luzio and Riggi, 1964). The “RE test lipid emulsion” was injected intravenously at a triglyceride dose of 25 mg/lOO gm body weight. Following the injection, five serial O.l-ml aliquots of whole blood were obtained from the tail at approximately 2-minute intervals and evaluated for total radioactivity. Blood radioactivity per milliliter blood, expressed as the percentage of t,he injected dose was plotted semilogarithmically against time in minutes ancl half-times t’tllS were determined. Liver, lung, and spleen tissue distribution of the labeled emulsion was determined on aliquots of organs taken 10 minutes after injection of the test colloid. Tissue uptake is expressed as the percentage of the injected dose per total organ (%ID/TO) and as the percentage of the injected close per gram tissue (%rro/gm) iDi Luzio and Riggi, 1964). The RES phagocytic function was also determined by the carbon-gel clearance technique as described elsewhere (T,ozzio, 1967a, 1967b). Animals w-cre injected intravenously with 8 me;/100 gm of body weight’ of colloidal carbon. Blood samples (50 ~1 each) were taken every 3 minutes for 15 minutes. Carbon concentrations were determined spectrophotometrically in the hemolyzed blood, plotted semilogarithmically against, time in minute,, q and half-times were calculated. The primary immune response to sheep erythrocytes was studied by injecting 1 ml of a 20% suspension of sheep erythrocytes intravenously. Animals were bled daily from days 5 to 7 and at 2 or 3-day intervals for 15 days after immunization. Serum samples were stored at -20°C until titrated. Serum complement was inactivated at 56°C for 30 minutes. Hemagglutinin and hemolysin titers were
PHAGOCYTOSIS
AKD
IMMUNE
RESPONSE
IN
GUNN
RATS
59
determined by serial twofold dilutions of inactivated serum as described elsewhere (Lozzio and Comas, 1969). Titers were expressed as the loga of the reciprocal of the highest dilution giving visible agglutination or complete hemolysis, respect.ively. The pattern of immunologic response was also evaluated by the plaque assay technique of Jerne et al. (1963). Rats were immunized as indicated previously and were sacrificed by total exsanguination at days 3, 6, and 12 after antigenic stimulation. The spleen was removed and weighed. Two pieces, weighing approximately 100 mg each, were gently teased and t.he cells suspended in 2 ml of Eagle’s medium. Nucleated cell counts were performed at this stage on each cell suspension. Since it was found that the number of cells in a piece of spleen tissue is proportional to its weight, the tot.al number of spleen cells was calculated on the basis of the average cell number found in two pieces of splenic tissue, multiplied by the total weight of the organ. This method has an error of no greater than lo%, and obviates the cumbersome and time consuming teasing of the whole spleen of the rat. The cell suspensions were diluted 1: 100 and 1:250. The number of spleen cells producing specific antibodies against sheep erythrocytes was determined by direct (19s) hemolytic plaque formation in agar gel, using duplicate aliquots (0.5 ml) of each dilution. The average of four determinations was expressed as plaque counts per million spleen cells, or per whole spleen. Microscopic examination was performed on specimens of the liver, spleen, lungs, bone marrow, lymph nodes, kidneys, and adrenal glands. Tissues were fixed in 10% phosphate buffered formaldehyde (pH 7.0) solution and processed by standard techniques. Sections were stained with hematoxylin-eosin and PAS. Iron was analyzed as indicated by Gomori (1953). Serum bilirubin concent.rations were determined by the diazo reaction. RESULTS The liver and spleen weights were similar in Wistar rats of both sexes (Table I). A significant (p < .OOl) enlargement of the liver was observed in jJ and jj Gunn rats of both sexes when compared with Wistar rats. Ninety per cent of the jj females also had significant (p < .OOl) splenomegaly when compared with other groups of rats studied. The kidneys appeared significantly (p < .Ol) enlarged in jj rats of both sexes. There was no difference in the weight of the lungs. Ovariectomy or splenectomy did not influence the weight of the liver in jJ and jj female Gunn rats. Splenomegaly was unchanged in ovariectomized jj animals. Bilirubin was not detectable in the serum of Wistar and jJ Gunn rats. A mean serum bilirubin concentration of 6.45 f 0.36 mg % was found in 80 jj animals of both sexes with a range of 5.2 to 9.0 mg %. The intravascular clearances (h/z) of the triolein 1311-labeled emulsion were nearly identical (13.1-14.6 minutes) in male and female Wistar rats (Table II). The removal of lipid emulsion was markedly enhanced in jj (5.7 minutes) and jJ (9.2 minutes) females of the Gunn strain when compared with either their male
60
LOZZIO.
M3CHdDO.
3KD
TABLE ORGAS
WEIGHTS
ANL)
Homozygous Male Female
Liver
10 10
3.32 3.GG
f f
25 24
4.09 4.58
2G 40
4.18 4.87
weight
Spleen
0.09a 0.08
Kidneys
Lungs
0.20 0.25
f f
0.01 0.01
0.M f O.BO f
0.02 0.03
0.74 0.73
* *
0.02 0.02
f 0.3-l III 0.30
0.24 0.26
* f
0.07 0.01
0.51. f O.GO i
0.008 0.01
0.83 0.78
zt 0.01 32 0.03
f f
0.25 0.37
z!z 0.01 + 0.01
0.61 O.F&
0.02 0.02
0.89 0.92
zt 0.02 f 0.03
Gmm
aMeanf
0.12 0.06
* f
1SE TllBLE CLEARAXE
AND
TISSUE
EMULSION”
II
I~ISTRIBUTION IN
WISTAR
OF AND
THE
No. of rats
Group
Liver 74, m/TO
Wistar Male
TEST
LIPID
R.\Ts uptake
r% ID)
Lungs
I % m/m
“RF:
GE.,.ITIKIZED
GUNN Organ
Female Heterozygous Male Female Homorygous Male FlXTl&
1 ATS
Gunn
Female
VASCULAR
GUKN
70 of body
of rats
Wist,ar Male Female Heteroxygous Male
LUZIO
I
OF WISTAR
I 0. Y
Group
DI
Spleen
70ID/TO
__w ID/‘:*
14.6 f l.4a 13.1 * 2.2
36.8 It 3.G 5.0 f 47.6 f 2.7 4.9 i
0.7 0.1
1.4 f 0.1 1.1 + 0.6
1.1 z!z 0.2 1.0 rt 0.1
3.9 * 0.4 8.3 + 0.6
7.3 l 15.3 f
16.4 f 9.5 *
47.5 zk 3.7; 4.5 f 50.3 * 4.1 7.0 f
0.4 0.5
1.5 f 1.7 f
1.1 i 1.3 f
4.0 * 0.3 4.8 * 0.2
6.5 zt 2.2 10.5 f 0.6
40.5 f 60.5 f
0.6 1.0
1.4 zk 0.1 1.5 i 0.1
0.6
2.7
Gunn 5.7 0.9
0.1 0.1
0.1 0.1
Gunn 14.4 zt 1.8 5.7 f 1.8
3.1 5.0
3.5 f 7.3 i
1.1 * 0.1 1.4 f 0.2
4.4 * 3.9 f
0.2 0.4
i.4 x.i
rt 0.3 i 1.7
counterparts, or wit’h Wistar rats. The increase in vascular clearance in female Gunn rats appears related, in part, to increased liver uptake as denoted by the %In/gm values. The uptakes of the lungs and spleen were unaltered in Gunn rats as determined on either a total organ basis or on the basis of %ID/gDl. The rate of carbon-gel clearance (Table III) was equivalent in Wistar rats, jJ animals of both sexes and jj male Gunn rats. In contrast, a mild but constant and significant (p < .OOl) acceleration of the rate of carbon clearance was observed in the majority of Jj female Gunn rats. The RES carbon phagocytic function was normal shortly (7 days) aft’cr splenectomy, while a 30-day period was necessary to achieve the normalizat’ion of phagocytosis in ovariectomixed jj animals. The same surgical procedure P did not alter phagocytoeis of colloidal carbon in i-J female animals. The appearance of the reticuloendothclial cells (RE) in different. organs of the Gunn rat resembles that of the RES under moderate stimulation. The Kupffer
PHAGOCYTOSIS
SNI)
IMMUNE
RESPONSE
TABLE IUS
C.\KHOK
PH.\GOCYTIC c;UNN
GUNN
RATS
61
III ACTIVITY
OF WIGT.IE
.LND
R .\TS NO.
of rats Wistar Male Female Heteruygous Malt Female Homo:.ygous Male Female Splenectomized Heterozygow Homozypous Ovariecton~ized Heterozygous Heterozjgu,ls Homozygotw IIomozygou~
IN
10 10
Days after surgery
-
Carbon clearance (t112, minutes)
12.8 12.3
* +
0.2ou 0.26
20 20
11.0 12.4
+ +
0.3-l 0.55
20 20
12.9 8.6
+ *
0.50 0.31
13.4 12.3
Zt 0.19 k 0.09
12.5 l-k.1 10.0 15.2
+ * f f
Gunn
GUMI
Female 10
i
10
7
10
7
10 10 10
30 i 30
0.10 0.16 0.21 0.16
cells appeared hyl)ertrophic (Fig. 2). The majority had a round or oval shape instead of the typical triangular or stellate shape characteristic of those cells in Wistar rats (Fig. 1) . The anatomical distribution of Kupffer cells was preserved. Phagocytoeis of carbon by Kupffer and other liver reticuloendothelial cells was proportional to their size and number. A markedly increased number of reticulum cells and macrophages was observed in the spleen of Gunn rats of both sexes as compared with the spleens of JYistar rats (Fig. 3). The picture in the red pulp was characterized by the presence of an abnormal number of active macrophages containing larger iron &ores than those of Wistar rats. In Gunn rats, hypertrophy of the endothelial sinus lining cells caused narrowing of the sinusoidal lumen. Numerous reticulum cells populated the hypertrophic splenic follicles (Fig. 4). The marginal zone was enlarged by the presence of an abnormal number of macrophages and reticulum cells. A marked increase of splenic carbon granulopexy was found when compared with JVirtar rats. A similar picture, characterized by an increased number of cells able to phagocytize carbon, was observed in the lungs, bone marrow, lymph nodes, adrenal glands, and kidneys. Natural antibodies to sheep erythrocyten were not found in t’he serum of the rats studied. The agglutinin and hemolysin levels in Wistar and Gunn rats attained a maximum titer 4-7 clays after antigenic stimulation, and no significant differences of the humoral antibody titers were observed between the two strains (Fig. 5 ) . As shown in Table IV, the peak of plaque-forming cells (PFC) occurred on
62
LOZZIO,
MACHADO.
AND
DI
LUZIO
The lxeswt~ stutlic>s in~lic:~tt~ t,hat ( lure r:its 11:/w hqmtonwgaly. :m alteration of the number ant1 :wtivit\of RES wlls, :mtl a tlin~inishetl numlwr of splenic antibody-forming ~11s n-htw coinpared with their lmrcwt strain, the Wistar rat. In addition, ij ft9wlcs niaiiifcstetl s.plent~niegwlpand both j.J ant1 jj femalw sl~owctl stimulation of RIM phagocytic actkit?- :w clc~tcrmiutd eithcxr I)\- “RN lipid emulsion” or carbon-gel methods.
PHAGOCYTOSIS
AND
IMMUNE
RESPONSE
IN
GUNN
FIG,. 3. Srction of the spleen from a female Wistnr rat marginal zone (MZ) and red pulp. (RP). H.E. X 200. FIN. 4. Section of the spleen from a homozygous frmnle t!!c splcnic follicles (SF) and enlargrmrnt of thrl mnrginal wt=n (RP). H.E. X 200.
?howin
g the
Gwm xon?
RATS
splenir
ant. s!:owing (MZ). The
63
follicles
(SF).
l~ypertrol~lly of wtl pulp is also
As a consecpence of the bilirubin transferase deficiency, jj Gunn rats have permanently high blood and tissue levels of bilirubin which is bound to albumin and other protein fractions (Housset et al., 1967 1. Since albumin-bound bilirubin exerts no harmful effects on mammalian cells iCowgcr et nl., 1965; Kikuchi, 1961) and some of the pathophysiologic changes described in this study were present in both j,J and jj animals, t’he increased amount’ of bilirubin was probably not the cause of the alterations noted. Bartonellosis can be ruled out as a cause of the splcnomegaly and the hematologic alterations previously described (Lozzio et nl., 1970) because the animals, on repeat,ed examination, were found to be frw of Rartonelln murk. Both Wistar
-
WISTAR
(26)
l
4
GUNN
(34)
HEMOLYSIN
234567 2
9
I
AGGLUTININ
(2
(5
PHAGOCYTOSIS
AND
IMMUNE
RESPONSE
IN
GUNN
RATS
65
The RES hyperfunction found in fcmalc Gum rats was associated with hepatomegaly. However, the hepatomcgaly doe.+ not seem to be the sole cause of the RES hyperfunction because jJ and jj malts had hepatomegaly and normal phagocytic function. Normal removal of carbon from the circulation was observed in Ii females 7 days after splenectomy or 30 days after ovariect’omy. Moreover, jj female Gunn rats have a syndrome of hypersplenism involving the erythrocytee. It is characterized by mild anemia and shortened erythrocyte survival time, caused by augmented splenic sequestration (Lozzio et nl., 1970). Therefore sex differences and splcnic hyperactivity remain as l)ossible factors in the pathogenesis of the anemia (Lozzio et nL.. 19701 and of the alteration of the RES phagocytic function. It has long been known that splcncctomy combined with ovariectomy creates the optimum conditions for the expcrimcntal blockade of the R,ES (Leites and Riabow, 1928). Recent studies inclicat,e that the eplee~~ is a significant RE organ 3s denoted by the decreawl clearance rate of the “RE lipid emulsion” in splenectomized rats (Patterson et nl., 1970). The mechanism by which the spleen influences RES phagocytic funct’ion is unknown. The effects of estrogens on the RES hare hecn recently reviewed (,VernonRoberts, 1969). RES act’ivity varies with the estrous cycle and falls after ovariectomy. Conversely, the administration of natural and synthetic estrogens produces hepatosplenomcgaly due to a striking hyperplasia of RE cells associated with hyperphagocytic activity (Vernon-Roberts, 1969). In t,hc present study, Gunn rat s were noted to have hypertrophy of liver macrophages and hel)atomegaly. Hyperplasia of splenic RE cells was found in all rats of the Gum strain, including those with normal RE phagocytic function. Studies demonstrating that splenectomy or ovariectomy in jj females suppress the hyperphagocytic function confirm previous observations on the role of the spleen in RES activity. At the same t)ime, they provide indirect evidence for the involvement of estrogen in RES activity even though ovariectomy did not affect splenomegaly in thwc rats. The fact that infertility occurs in all jj female Gunn rats further suggests a disturbance of sex hormone metabolism. Although splenic follicles alqwarcd hypertrophic and hypcrplasia of the reticulum cells and of the macrophages was present in Gunn rats, fewer antibody form’ng cells were found in the spleens of Gunn rats than in the spleens of Wirtar rats. This finding indicates that the proliferation of certain cell lines does not neces::arily make available a grcatcr number of cells able to react with the antigen. In fact, the hypcrtrophy of the lymphoirl follicles and the RE hyperplasia would sew1 to bc detrimental to iInmunocompt~t~~tlt cells since there were more splcnic cclla after antigenic stimulation in Gunn than in Wistar rats, while the number of plaque-forming cells was diminished. Since the splecm is the principal organ of hemolysin formation in the rat (Rowley, 1950) it appears that the number of splenic antibody-forming cells obs;cr\-ed in Gunn rats was sufficient rats to maint’ain similar agglutinin and hemolysin titers to those of Wistar throughout, the primary immune response. It is also probable that other areas of the lymphopoietic system compensate for the diminished number of antibodies formed in the spleen of Gunn rat’s.