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Hematologic and serologic studies in 6-sulfanilamidoindazole arthritis.

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68 1
HEMATOLOGIC AND
SEROLOGIC STUDIES IN
6-SULFANILAMIDOINDAZOLE
ARTHRITIS
MORGAN L. MILLER, CECIL 0. SAMUELSON, and JOHN R. WARD
6-sulfanilamidoindazole (6-SA1), an antibacterial
sulfonamide, is known to cause an acute, self-limited inflammatory reaction in the hind paws of older rats. Administration of 6-SAI results in hyperfibrinogenemia
which precedes the onset of clinical arthritis. The hyperfibrinogenemia is associated with the prolongation of
dilute blood clot lysis times, appearance of heparinprecipitable fibrinogen, elevations in fibrin degradation
products, and complement titers and serum electrophoretic pattern changes that consist of decreased albumin
concentrations and increased beta globulin concentrations.
Alterations in 6-SAI medicated young rats are less
marked and require higher doses.
In 1964 Mielens and Rozitis (1) described an
acute, self-limited inflammatory reaction in the joints
and periarticular tissues of the hind paws of older rats
medicated orally with an antibacterial sulfonamide, 6sulfanilamidoindazole (6-SAI). Subsequent studies on
6-SAI arthritis have shown that the inflammation is apparently independent of high blood sulfonamide conFrom the Division of Arthritis, Department of Internal Medicine, University of Utah College of Medicine, Salt Lake City, Utah.
Supported in part by grants from the Kroc Foundation and
Arthritis Foundation Clinical Research Center.
Morgan L. Miller, Ph.D.: Post-doctoral Fellow (present address: Department of Microbiology, University of Alabama): Cecil 0.
Samuelson, M.D.: Assistant Professor of Medicine: John R. Ward,
M.D.: Professor of Medicine and Chief, Arthritis Division.
Address reprint requests to Cecil 0. Samuelson, M.D., Arthritis Division, Department of Internal Medicine, University of Utah
College of Medicine, Salt Lake City, Utah 84132.
Submitted for publication January 1 1 . 1978: accepted in revised form March 13. 1978.
Arthritis and Rheumatism, Vol. 21, No. 6 (July-August 1978)
centrations, crystal formation in tissues, kinin formation
through activation of the Hageman factor, immediate
and delayed hypersensitivity reactions, induction of an
anaphylactoid-like reaction, or provocation of a latent
mycoplasma infection (2,3). Further, 6-SAI arthritis is
not altered by induction of liver enzymes (4),treatment
with histamine, bradykinin and serotonin depleting
agents ( 5 ) , or by induction of alloxan diabetes (6).
As yet it has not been possible to develop a
reasonable hypothesis to explain 6-SAI arthritis. However, recent studies in this laboratory have shown that
feeding 6-SAI to aged rats is associated with hyperfibrinogenemia which antedates the onset of clinical disease. Thus it was considered useful to further define this
feature of the disease. The purpose of this communication is t o report studies on: 1 ) the relationship between
histological and clinical changes in the joints to changes
in plasma fibrinogen concentration, volume of packed
red blood cells, white blood cell counts, and total blood
sulfonamide concentrations; 2) blood clotting activity,
serum complement levels, and changes in serum electrophoretic patterns in 6-SAI medicated rats; and 3)
possible roles of aging, inflammation, and the pituitary
gland in 6-SAI induced hyperfibrinogenemia.
MATERIALS AND METHODS
Animals. Holtzman strain male albino rats were used
in all experiments. Except when specified, animals employed in
these experiments weighed 480 to 580 grams.
6-SAI Dosage and Route of Administration. 6-SAI was
supplied in a powder form by Zigurd E. Mielens of the Sterling
Winthrop Research Institute, Rensselaer, New York. For oral
MILLER ET AL
682
administration, 6-SAI was suspended in 1% methylcellulose,
150 mg/ml, and was administered via gavage tube. For subcutaneous injections, 6-SAI was dissolved in 100% propylene
glycol, 75 mg/ml. Dosages of 250 mg/kg body weight were
employed in all experiments, except as specified in experiments
with young rats where 500 mg/kg body weight was used,
Blood Sulfonamide Concentrations. Sulfonamide levels
were determined on blood filtrates following acid hydrolysis
by the method of Bratton and Marshall (7), and the results
were expressed as total blood sulfonamide concentration.
Arthritis Scoring. The technique for scoring inflammation was adopted from the arthrogram described by Sabin and
Warren (8). The arthritis score was obtained immediately
prior to sacrifice, All animals were sacrificed 24 hours after the
last feeding.
Blood Studies. Blood from the abdominal aorta was
withdrawn in plastic syringes with 20 gauge needles. The following studies were done:
Plasma Jibrinogen concentration. Blood was collected
in 38% sodium citrate, 1.0 ml blood per 0.01 ml anticoagulant,
and the plasma fibrinogen concentrations were determined by
the method of Ellis and Stransky (9). This method is a rapid,
thrombin catalyzed, indirect spectrophotometric method
which has been evaluated only in human plasma. It was routinely used to measure fibrinogen concentrations in all experiments. To confirm that the material in rat plasma as measured
by this technique is fibrinogen, fibrinogen concentrations were
also determined by the technique of Goodwin (10). This is a
direct method for measuring fibrinogen that depends upon
precipitation of the protein by sodium sulfite. To determine
whether sodium sulfite precipitates proteins other than fibrinogen in the plasma of rats, the reagent was also added to the
sera of these animals. The plasma samples employed in this
comparative study were obtained from groups of rats sacrificed following a single intravenous injection of 100 pg of
endotoxin, 5 oral administrations of methylcellulose, and 5 or
7 feedings of 6-SAI.
Fibrinogen concentrations in the plasma of all groups
of rats were not significantly different as determined by the two
techniques (Table I ) . Furthermore, sodium sulfite did not
precipitate rat serum.
Table 1. Comparison of the Ellis-Stransky and Goodwin Techniques
for Measuring Fibrinogen Concentration in Rat Plasma
Mean Plasma Fibrinogen
Concentration mg/100 ml (Rangej
Treatment
~
~
No.
of Rats
~
10
Endotoxin IV X I
10
6-SAI oral X 5
15
6-SAI oral X 7
24
=
> 0.05
Goodwin
~~
1% methylcellulose X 5
*P
Ellis-Stransky
343
(236-641 )
525
(357-693)
1309
(578-2205)
1450
(9 19- 1900)
370*
(320-61 5)
5008
(357-620)
I199*
(690-1750)
1315*
(870-1 594)
Dilute blood clot iysis time (DBCLT). DBCLT, a measure of plasminogen activation, was determined in duplicate
by the method of Gallimore, et al. ( 1 I).
Fibrin degradation products (FDPJ. Serum F D P was
determined by the staphylococcal clumping test (12). The standard in these experiments was normal rat plasma diluted to
100 p g fibrinogen per ml, and the serum F D P was expressed as
pg fibrinogen equivalents per ml.
Heparin-precipitable Jibrinogen (HPF). HPF is an altered form of fibrinogen in heparinized plasma that forms a
flocculent or stringy precipitate in the cold which redissolves
upon warming (13). To assay for HPF, 4.0 ml of blood were
added to 400 units of heparin (sodium heparin, 1000 units/ml,
Upjohn). The plasma was separated by centrifugation, placed
in an ice bath, and observed for the presence of a precipitate 4
hours later.
Serum complement titer. A 2% suspension of sheep red
blood cells was sensitized with two 100% units of hemolysin
(Grand Island Biological Company) in barbital buffered saline, and the complement titers in hemolytic units per ml were
determined by the technique of Carpenter (14).
Total iron binding capacity (TIBC). The TIBC was
determined on blood collected in iron free tubes by the method
of Ramsay (15).
Serum electrophoretic pattern. Total protein concentrations were determined on 0.2 ml of sera in a Bausch and Lomb
refractometer standardized against distilled water. Serum
components were separated on 10 microliter samples applied
to a cellulose membrane in a buffer-filled electrophoresis cell
(Beckman). The membranes were fixed and stained with ponceau S and the concentration of serum components determined in a Model R-110 Microzone Densitometer as described
by Beckman Instruments, Inc.
White blood cell ( W B C ) counts and volume of packed
red blood cells ( VPRC). The total and differential WBC counts
and VPRC were determined by standard methods used for
hematological measurements.
Platelet counts. Platelet counts were performed by the
technique of Piette and Piette (16).
Sequential Studies on Arthritis, Blood Sulfonamide
Concentration, Plasma Fibrinogen Concentration, VPRC,
WBC, and Platelet Counts. Rats were divided into a control
group and 4 treatment groups with 8 to 10 animals per group.
Control animals were killed 24 hours after 5 administrations of
requisite volumes of methylcellulose, and treatment group rats
were killed 24 hours after 2, 3, 5, or 7 administrations of 6SAI. After killing, histologic studies were done on 6 to 10
randomly selected control animals and 6 of 8 treatment group
animals that had received 2 or 3 drug administrations. The
gastrointestinal tracts of these animals were examined for lesions.
Plasma fibrinogen concentrations, blood sulfonamide
concentrations, total and differential WBC counts, and VPRC
were determined in all animals. Platelet counts were determined on control animals and in rats killed after 5 and 7
administrations of 6-SAI.
Sequential Studies on Fibrinogen Concentration, Fibrinolytic Activity, Serum Complement Levels, Serum Electrophoretic Patterns, and Total Iron Binding Capacity. Twentyfive rats were divided into 4 treatment groups and 1 control
group with 5 animals per group. Treatment group animals
received either 2, 3,5, or 8 consecutive daily administrations of
STUDIES IN 6-SAI ARTHRITIS
6-SAI while control animals received 8 administrations of
requisite volumes of 1% methylcellulose. Immediately following killing, the fibrinogen concentration and DBCLTs were
determined in all animals while HPF was assayed i n the
plasma of control animals and in rats receiving 5 and 8 drug
administrations. Sera for the FDP, total hemolytic complement activity, and electrophoretic patterns were frozen at
-7O"C, and these determinations were performed on all medicated and control animals simultaneously at completion of the
experiment.
Because the liver synthesizes the iron binding protein
transferrin, which is distributed in the hepatocytes of rats in a
manner similar to that of fibrinogen and albumin (l7),transferrin concentrations were estimated by the total iron binding
capacity on the above control animals and in rats receiving 8
administrations of 6-SAI.
Comparative Study on Arthritis, Total Blood Sulfonamide, and Plasma Fibrinogen Concentration in Young and Aged
Rats. Because it is difficult to induce arthritis in young rats (1,
2, 18) and the blood sulfonamide concentrations in young
animals are less than in older animals receiving 6-SAI in
dosages of 125 mg/kg body weight (4),the effect of differing
dosages of 6-SAI on arthritis, blood sulfonamide levels, and
plasma fibrinogen concentration in young and aged animals
was studied. In these experiments animals weighing either 100125 or 500-550 grams received 6-SAI in dosages of 250 or 500
mg/kg body weight daily for 5 consecutive days and were
killed 24 hours after the last feeding.
Effect of Hypophysectomy on 6-SAI-Induced Hyperfibrinogenemia. Because ACTH (19) and somatotrophin (20)
stimulate fibrinogen production in experimental animals, the
effects of hypophysectomy on fibrinogen production in 6-SAI
treated rats were studied. Previous studies have shown that
hypophysectomized rats are extremely labile and die from
stress during gavage feeding (21). Therefore, animals in these
experiments received 6-SAI subcutaneously. Seventeen rats
(Holtzman Company, Wilmington, Maine) were hypophysectomized, 7 animals were sham hypophysectomized, and 7
unoperated rats served as controls. All animals were maintained on an enriched diet of ground meat and orange juice.
On the fourth post-surgery day, 4 hypophysectomized rats
were killed for determination of control plasma fibrinogen
concentrations; 1.0 ml of cardiac blood was withdrawn from
sham hypophysectomized and unoperated control animals for
similar determinations. Corticosterone (lot #Y3149, Schwarz/
Mann, Orangeburg, New York) was dissolved in 100%propylene glycol, 10 mg/ml, was administered subcutaneously starting on the fifth post-surgery day to the remaining hypophysectomized rats in doses of 5 my/kg body weight daily for 5
consecutive days. Sham hypophysectomized and unoperated
control animals received requisite volumes of propylene glycol. On the eighth and ninth post-surgery days (fourth and
fifth days of administration of corticosterone or propylene
glycol), 9 hypophysectomized and 7 sham hypophysectomized
rats received 250 mg/kg 6-SAI subcutaneously, and 7 control
animals received propylene glycol. Twenty-four hours after the
last treatments, all animals were killed and plasma fibrinogen
concentrations determined.
Effect of Methylprednisolone on 6-SAI-Induced Arthritis and Hyperfibrinogenemia. Thirty-two rats were divided into
3 treatment groups and 1 control group with 8 animals per
group. Treatment group animals received 6-SAI daily for 7
683
days while control animals received 1% methylcellulose.
Methylprednisolone sodium succinate, 40 .mg/ml (SoluMedrol, Upjohn), was diluted in phosphate buffered saline,
pH 7.40,to a concentration of 5 mg/ml and was administered
orally to treatment group animals for 8 days in dosages of 2.5,
3.5, or 5.0 mg/kg, respectively, every 12 hours starting one day
before initiation of 6-SAI treatments.
RESULTS
Sequential Studies on Arthritis, Plasma Fibrinogen Concentration, Blood Sulfonamide Concentrations,
VPRC, WBC, Counts, and Platelet Counts. Plasma fibrinogin concentrations were elevated in all groups of
sulfonamide medicated animals (Table 2). However, arthritis appeared in 1 o f 8 rats receiving 2 sulfonamide
feedings a n d in 2 of 10 rats receiving 3 drug administrations. Microscopic studies on the joints demonstrated
significant lymphocytic infiltration with proliferation of
t h e synovium in 2 of 6 rats killed after 2 drug administrations and in 4 o f 6 animals killed after 3 drug administrations.
There were no gross lesions in the gastrointestinal tracts of any sulfonamide treated animals.
Mean total blood sulfonamide concentrations
varied between 3.05 and 3.98 mg/100 ml in rats receiving 2 or more 6-SAI administrations.
Total white blood cell counts on control animals
a n d in all groups of sulfonamide medicated animals
ranged from 12,367 to 15,975/mma. Differential white
cell counts on blood of control animals revealed 8 to
25% polymorphonuclear cells ( P M N s ) and 75 to 92%
mononuclear cells, whereas blood of medicated animals
contained from 36 t o 45% P M N s a n d 5 5 to 64% mononuclear cells. Platelet counts in both control animals and
in animals killed after 5 a n d 7 sulfonamide feedings
ranged between 900,000 a n d 1,744,000/mm3. Only animals receiving 7 sulfonamide treatments had a significant decrease in mean V P R C (Table 2).
Sequential Studies on Fibrinogen Concentrations,
Fibrinolytic Activity, Serum Complement Levels, Serum
Electrophoretic Patterns, and Total Iron Binding Capacity. Increases in plasma fibrinogen concentrations were
observed in all groups of 6-SAI medicated rats (Table 3).
Although arthritis did not appear in rats receiving 2
sulfonamide treatments a n d in only 1 rat receiving 3
sulfonamide treatments in this experiment, plasma fibrinogen concentrations were elevated over that of control animals by approximately 50% a n d 150%. respectively. Animals receiving 5 a n d 8 d r u g treatments had
fibrinogen concentrations greater than 1400 mg/100 ml
a n d mean arthritis scores of 8 a n d 40, respectively.
There was a direct relationship between the dilute blood
MILLER ET AL
684
Table 2. Arthritis, Fibrinogen Concentration. Volume of Packed Red Blood Cells, and Total Blood
Sulfonamide Concentration in 6-SAI Treated Aged Rats
~
No. of Rats
With Total
Arthritis No.
Treatment
I % methylcellulose X 5
0
10
6-SA1 X 2
1
8
6-SAI X 3
2
10
6-SAI X 5
8
10
6-SAI X 7
10
10
*P
tP
=
=
~~~~~
Mean Plasma
Fibrinogen
Concentration
mg/100ml
(Range)
Mean VPRC
m1/100 ml
(Range)
343
(236-64 I )
580*
(420-725)
695t
(550-930)
1276t
(8 12- 1600)
1327t
( 1029- I 502)
47.0
(43.5-50.0)
46.0
(45.0-47.5)
46.0
(45.0-50.0)
47.0
(45-50)
39.5*$
(36.5-44.0)
~
~~
Mean Total
Blood Sulfonamide
Concentration
mg/100 ml
(Range)
0
3.05
(2.90-3.30)
3.92
(3.28-4.40)
3.56
(3.04-4.00)
3.98
(3.50-4.60)
< 0.05
< 0.01
$ The only group of 6-SAI medicated animals in which there was a significant decrease in mean VPRC.
clot lysis time and the number of 6-SAI administrations.
Administrations of 6-SAI resulted in a 2 to 6-fold increase in concentrations of fibrin degradation products
with the highest concentrations appearing in the sera of
groups of animals with arthritis. No heparin precipitable fibrinogen appeared in the plasma of control animals; however, it was readily demonstrable in the
plasma of rats receiving 5 and 8 drug administrations.
Changes in serum electrophoretic patterns in the
sulfonamide treated animals were characterized by progressive decreases in albumin concentrations and increased beta globulin concentrations without changes in
total protein concentrations (Table 4). Total hemolytic
complement titers of 272 hemolytic units were found in
the sera of control animals. Increased titers resulted
from 3 drug administrations, and peak titers approxi-
Table 3. Arthritis. Fibrinogen Concentration. and Fibrinolytic Parameters in 6-SAI Medicated Rats
No. of Rats
Treatment
Mean Plasma
Fibrinogen
Mean
With Total Conc. mg/lO ml DBCLT/min.
(Range)
(Range)
Arthritis No.
1% methyl-
0
5
0
5
cellulose X 8
6-SAI X 2
6-SAI
X 3
1
5
6-SAI X 5
5
5
6-SA1 X 8
5
5
* P = < 0.05
= < 0.01
$ P = < 0.02
tP
5 ND
=
Not done
338
(305-409)
529*
(431-630)
874t
(7 14- 1050)
1447t
( I 260-1785)
1405t
(1 155-1680)
51
(47-70)
81
(67-100)
948
(77-108)
I20$
( 129-240)
206$
( 120-240)
Mean FDP
pg/ml
(Range)
28
(1 2-50)
Heparin
Precipitable
Fibrinogen
-
83
ND§
67
(25- 100)
158,
( 100-200)
160*
( 100-200)
ND
(SO- loo)
+
+
STUDIES I N 6-SAI ARTHRITIS
685
Table 4. Serum Protein Concentration. Serum Complement Titers. and Total Iron-binding Capacity in 6-SA I Medicated Rats
Serum Protein Concentration g/100 ml
Total
Protein
6.31
(6.20-6.40)
6.32
(6.10-6.60)
6.42
(6.05-6.70)
6.40
(6.00-6.80)
5.89
(5.25-6.25)
Treatment
I% methylcellulose X 8
6-SAI X 2
6-SAI X 3
6-SAI
X 5
6-SAI X 8
*P
tP
Mean C' Hemi Mean TIBC
Units ml
ng/100 ml
(Range)
(Range)
Albumin
Alpha 1
Globulin
Alpha 2
Globulin
Beta
Globulin
Gamma
Globulin
2.80
(2.63-3.05)
2.48
(2.25-2.66)
2.25*
( I .79-2.90)
1.97t
(1.80-2.05)
1.44t
(1.29- I .59)
1.81
(1.54-2.25)
1.52
( I .41-1.69)
I .59
( I .35- I .79)
2.21
(1.94-2.50)
1.84
(1.40-2.10)
0.79
(0.60-1.03)
0.75
(0.56-0.95)
0.77
(0.60-0.87)
0.9 I
(0.77-1.20)
0.54
(0.15-0.76)
0.59
(0.39-0.67)
1.00t
(0.87- I .09)
1.20t
(0.98- 1.43)
0.94$
(0.66- 1.20)
l.50t
(1.37-1.77)
0.34
(0.14-0.44)
0.57
(0.54-0.61 )
0.48
(0.26-0.71 )
0.36
(0.26-0.46)
0.59
(0.37-1.02)
272
(250-286)
3054
(286-334)
392$
(334-500)
766
(680-830)
soot
(500-500)
500t
(500-500)
689s
(594-783)
< 0.05
< 0.01
$ P = < 0.02
4 P = > 0.05
=
=
mately twice those of control levels appeared in the sera
of arthritis rats receiving 5 and 8 sulfonamide feedings.
There were no changes in mean total iron binding capacity.
Comparative Study on Arthritis, Total Blood Sulfonamide, and Plasma Fibrinogen Concentrations in
Young and Aged Rats. Clinical arthritis appeared in only
1 young rat receiving 6-SAI in dosages of 500 mg/kg
and in no young animals receiving the 250 mg/kg dosages (Table 5). The majority of aged animals receiving
similar treatments developed arthritis. Fibrinogen concentrations in young rats were significantly elevated only
in the plasma of animals receiving 6-SAI in dosages of
500 mg/kg. Sulfonamide concentrations in the blood of
aged rats receiving 6-SA1 in dosages of 500 mg/kg and
250 mg/kg were 316% and 78% higher than those in
young animals.
Effect of Hypophysectomy on Plasma Fibrinogen
Concentrations. Mean fibrinogen concentration in the
Table 5. Arthritis, Plasma Fibrinogen, and Total Blood Sulfonamide Concentration in Young and Aged
Rats Receiving 6-SAI
No. of Rats
Treatment
Weight
With Total
Arthritis No.
Mean Total
Blood Sulfonamide
Conc. mg/100 ml
(Range)
Mean Plasma
Fibrinogen Conc.
mg/100 ml
(Range)
100-125
0
14
0
500-550
0
8
0
100-125
0
10
2.39
(0.90-3.41 )
308
(300-483 )
298
(215-420)
413t
( I 30-641 )
500-550
7
10
100-125
I
I1
4.25*
(3.75-5.80)
1.76
(0.40-3.12)
I142*
(806-1480)
455*
(210-725)
500-550
6
7
7.32"
(3.29-11.36)
(1 28 1-1 680)
I% methylcellulose X 5
6-SAI
250 mg/kg X 5
6-SAI
500 mg/kg X 5
*P
tP
=
=
< 0.01
> 0.05
1488*
MILLER ET AL
686
plasma of 4 hypophysectomized rats killed on the seventh post-surgery day was similar in concentration to
that of sham hypophysectomized and control rats
(Table 6). Several of the hypophysectomized sulfonamide challenged animals were near death when killed.
None of the animals developed arthritis. Mean plasma
fibrinogen concentration of hypophysectomized and
sham hypophysectomized rats following 2 drug administrations exceeded that of control rats challenged with
propylene glycol.
Effect of Methylprednisolone on 6-SAI-Induced
Hyperfibrinogenemia. Six of 8 rats receiving methylprednisolone in dosages of 5.0 mg/kg developed arthritis, whereas no rats receiving either 7.0 or 10 mg/kg
doses developed arthritis (Table 7). Plasma fibrinogen
concentrations in non-arthritis rats receiving 7 mg/kg
doses of methylprednisolone were significantly elevated
over control concentrations.
DISCUSSION
Both 6-SAI disease and “adjuvant disease” are
peculiar to rats. Adjuvant disease is best induced in
young but not aged animals (22), and the arthritis results from a delayed response to disseminated constituents of mycobacteria (23). 6-SAI or one of its metabolites initiates inflammation.
Because these animals received large daily dosages of 6-SAI, total blood sulfonamide levels were determined. Results of these studies demonstrate that blood
sulfonamide levels are low. Indeed, the mean blood
sulfonamide concentrations in any group of aged rats
receiving 6-SAI in dosages of 250 mg/kg body weight
Table 6 . Effect of Hypophysectomy on Plasma Fibrinogen
Concentration of 6-SAI Medicated Rats
Pretreatment
Treatment
No.
Rats
Propylene glycol X 2
7
Sham hypox 6-SAI X 2
7
Hypox corticosterone X 5
6-SAI X 2
9*
Post-treatment
Fibrinogen Conc. Fibrinogen Conc.
mg/100 ml
mg/100 ml
(Range)
(Range)
362
(273-473)
358
(326-415)
367
(315-415)
44 1
(368-557)
I266t
(966-1 680)
1369t
(967-1680)
* All animals lost between 40 and 80 grams body weight and 9 of 13
animals survived.
t P = < 0.01
never exceeded 4.60 mg/100 ml. These drug levels are
much lower in concentration than blood levels of clinically employed antibacterial sulfonamides which are required for optimal therapeutic effects (24). Results of
these comparative studies on differences in blood sulfonamide levels in aged and young rats confirm those of
Hirschelman et al. (4) who reported that blood sulfonamide levels following 6 oral administrations of 6-SAI in
dosages of 125 mg/kg body weight were 50 to 100%
higher in old rats, These same investigators reported
that induction of hepatic enzymes with phenobarbital
did not influence either blood sulfonamide concentrations or intensity of arthritis in either young or aged
animals. Lower blood sulfonamide levels in young animals could result from less intestinal absorption or more
rapid excretion of the drug.
Results of these studies on the cellular components of the blood, together with those from a previous investigation (2), demonstrate that 6-SAI administration, like adjuvant injection, induces changes in the
neutrophil-lymphocyte ratios. Leukocytosis (25) and
anemia (26) are prominent features of adjuvant disease
and precede the onset of arthritis. A “tendency” to
leukocytosis and anemia has been reported in 6-SAI
disease (18). In the current investigation leukocytosis
was not observed. Furthermore only a mild anemia was
observed in one group of arthritic animals receiving
large numbers of 6-SAI feedings, whereas other groups
of similarly medicated animals were not anemic.
Elevation in plasma fibrinogen concentration
and cellular infiltrations of the paws of rats is an early
response to 6-SAI feedings and adjuvant injections.
However, the relationship of hyperfibrinogenemia and
cellular infiltrates in rats receiving 6-SAI orally remains
to be fully explained. The hyperfibrinogenemia does
precede microscopic evidence of inflammation. The histological changes in rats without clinical arthritis consisted of lymphocytic infiltration of the synovium with
synovial proliferation and were not observed in all animals. Because 6-SAI was administered orally, the gastrointestinal tracts were visually examined. Elevations in
plasma fibrinogen concentration could not be explained
by gross lesions in the stomach or intestines, but failure
to demonstrate such lesions does not exclude the possibility of microscopic lesions. Since 6-SAI is a potent
antibacterial sulfonamide it is possible that orally rnedicated animals develop an abnormal intestinal flora with
passage of bacterial products into the blood which in
turn stimulate fibrinogen production and inflammation.
Indeed, pigs fed a protein rich diet containing fish meal
developed within one week an abnormal intestinal mi-
STUDIES IN 6-SAI ARTHRITIS
crobial flora, as evidenced by a significant increase in
numbers of atypical Clostridium perfringens type A (27).
Increased erythrocyte sedimentation rates and aseptic
swollen peripheral joints appeared concomitantly with
changes in intestinal flora. However, this mechanism is
unlikely since none of 8 different antibacterial sulfonamides were arthritogenic in aged rats (18).
Administrations of 6-SAI were associated with
prolongation of the dilute blood clot lysis time and
appearance of fibrin degradation products and heparinprecipitable fibrinogen. Lengthening of the lysis time in
the hyperfibrinogenemic animals could result from an
increased quantity of fibrin to be lysed before complete
dissolution of the clot and not from a reduction in
concentration of plasminogen activator. Although the
lysis times were elevated in these experiments, they were
much less than those reported in the blood of hyperfibrinogenemic rats following subcutaneous challenge
with turpentine (1 1). The alpha-2-globulin fraction contains a plasmin inhibitor (28). This fraction is not elevated in 6-SAI disease but is in other types of inflammation (24,29). Thus, the comparatively shorter lysis times
observed in these experiments may be explained in part
by a relative deficiency of plasmin inhibitors, especially
when compared to the adjuvant model of inflammation.
Fibrin degradation products are derived from
fibrinogen and/or fibrin proteolysis (30). They may be
detected in some circumstances even when the consumption of humoral and cellular clotting factors cannot be
demonstrated. Fibrin degradation products in the sera
of aged control rats in these experiments were much
higher than those reported in younger control rats as
determined by the staphylococcal clumping assay (31).
The further elevation in concentration of fibrin degradation products and plasma fibrinogen in rats with 6-SAI
arthritis remains to be explained. The concentration of
fibrinogen in the plasma reflects the balance between the
rate of synthesis and rate of destruction. Because the
liver can rapidly generate fibrinogen, an episode of intravascular coagulation is followed by compensatory
hyperfibrinogenemia, as well as elevated fibrin degradation products which characterize the over-compensated
intravascular coagulation fibrinolysis syndrome (32).
There are other syndromes in which hyperfibrinogenemia and elevated fibrin degradation products
coexist without direct evidence of a preceding episode of
- intravascular coagulation. It has been reported that
both elevated fibrinogen concentrations and fibrin degradation products appear in the blood of rats following
a single subcutaneous injection of turpentine (1 1). Patients with rheumatoid arthritis have elevated concen-
687
Table 7. Effect of Methylprednisolone on 6-SAI-induced Arthritis
and Hyperfibrinogenemia
~~
No. of Animals
Treatment
With Total
Arthritis No.
I % methylcellulose X 8
0
8
6-SAI X 7 methylprednisolone
5 mg/kg X 8
6-SAI X 7 methylprednisolone
7 mg/kg X 8
6-SAI X 7 methylprednisolone
10 mg/kg X 8
6
8
0
8
0
8
*P
tP
$P
=
=
=
~~
Mean Plasma
Fibrinogen Conc.
mg/100ml
(Range)
304
(23 1-399)
I168*
(326- 1800)
9647
(294-1712)
4984
(294-1124)
< 0.01
< 0.02
> 0.05
trations of intravascular and extravascular fibrinogen.
The increased fibrinogen concentrations are secondary
to increased hepatic synthetic rates which seem to correlate with the severity of the disease process (33). In
addition to elevated fibrinogen concentrations, there are
significant elevations in levels of fibrin degradation
products in the sera of rheumatoid patients (34,35). It
has recently been suggested that fibrin degradation
products in hyperfibrinogenemic blood are derived from
either fibrinogen proteolysis, which maintains a dynamic equilibrium with increased fibrinogen synthesis
(35), or alterations of fibrinogen by interaction with
histones, lysosomes, or other enzymes released from
cells unassociated with intravascular coagulation (36).
Heparin precipitable fibrinogen is a complex
composed of fibrin degradation produces, intermediates
in the conversion of fibrinogen to fibrin, and possibly
other plasma proteins (37) and is detected in the blood
of humans by ethanol gelation and protamine sulfate
precipitation tests. Heparin precipitable fibrinogen has
been found in the plasma of patients with rheumatoid
arthritis, rheumatic fever, and systemic lupus erythematosus (38).
Changes in the serum electrophoretic patterns in
sulfonamide medicated rats preceded the onset of gross
arthritis and resembled those of adjuvant disease except
that alpha-2 globulin concentration is elevated in that
model of inflammation (24,30). Although different proteins, including transferrin, and several of the components of complement migrate with the beta-globulin
fraction, it is likely that increases in this fraction are
nonspecific. There was no increase in the total iron
binding capacity and peak levels of total hemolytic com-
MILLER ET AL
68 8
plement activity did not parallel increases in beta-globulin levels. The hypercomplementemia is of interest since
hemolytic complement activity is also elevated in adjuvant disease (29), rheumatoid arthritis (39,40), and
rheumatic fever (41) a n d may be another “acute phase”
reactant accompanying widespread inflammatory processes.
Despite treatment with corticosterone, the subcutaneous injection of 6-SAI severely stressed the hypophysectomized animals. It was thus necessary to kill
the animals after two drug administrations prior to completing the planned experimental protocol. Even after
two treatments with 6-SAI the hyperfibrinogenemia produced was impressive. These data suggest that fibrinogen production in sulfonamide medicated animals is
not dependent on pituitary hormones.
The demonstration that significant hyperfibrinogenemia in 6-SAI medicated rats by both the oral
and subcutaneous routes precedes gross and microscopic arthritis suggests that the elevated plasma fibrinogen concentrations in this experimental model a r e not
secondary to gross inflammation of peripheral joints.
T h e latter observation is in direct contrast t o that of Sigg
et al. (18) who reported that concomitant oral administrations of hydrocortisone (5.0 mg/kg) a n d 6-SAI (I25
mg/kg) suppressed both inflammation and fibrinogen
synthesis .
The above reported abnormalities associated
with 6-SAI administration are interesting, b u t they have
not yet been linked in such a way a s t o contribute t o the
understanding of the pathogenesis of 6-SAI arthritis.
Further studies of 6-SAI metabolism will be necessary
in young and aged rats. Fibrinogen synthesis, conversion, and fibrin degradation will require more detailed
investigation. Nonetheless, the model is unique a n d
merits further inquiry to understand its mechanism
as a potentially new form of joint inflammation.
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