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The migration of lymphocytes across specialized vascular endothelium
CELLULAR
IMMUNOLOGY
66,407-422
The Migration
IV. Prednisolone
(1982)
of Lymphocytes across Specialized Vascular Endothelium
Acts at Several Points on the Recirculation of Lymphocytes JOSEPHINE
Pathways
H. Cox AND W. L. FORD
Experimental Pathology Laboratory, Department of Pathology, Stopford Building, University of Manchester. Manchester MI 3 9PT, United Kingdom Received November 4, 1981: accepted November 23, 1981 Radioactively labelled thoracic duct lymphocytes from syngeneic rat donors were injected iv into recipients which had been given a continuous iv infusion of prednisolone at 1 mg/hr for 15-18 hr previously. The tissue distribution and recirculation into lymph of the labelled lymphocytes were compared quantitatively in the prednisolone-treated and control recipients by scintillation counting and autoradiography. The most prominent effect of prednisolone was to retard recirculating lymphocytes within the tissues to which they are normally distributed by the blood, namely the bone marrow, the spleen, and the lymph nodes.Although lymphocyte traffic was almost completely frozen by prednisolone, recirculating lymphocytes were not killed. A secondeffect of prednisolone was to impair the influx of lymphocytes from the blood into lymph nodes. Different groups of lymph nodes varied in the extent to which prednisolone inhibited the entry of lymphocytes, and previous antigenic stimulation completely exempted lymph nodes from this inhibition. Lymphocytes took a longer time to cross the walls of high endothelial venules in the lymph nodes of prednisolone-treated rats. A third effect of prednisolone was to increase the rate at which lymphocytes entered the bone marrow from the blood by crossing sinusoidal endothelium.
INTRODUCTION A great deal of work over the past three decades has established that the administration of corticosteroids may produce (i) suppression of immune responses ( 1, 2); (ii) destruction of large numbers of lymphocytes particularly in the thymus and germinal centres (3, 4), and (iii) a deficit of lymphocytes in the blood, as well as many other effects. Lymphocytopenia following large doses of corticosteroids has been described in man (5), mice (6), rats (7), and guinea pigs (8). Recent work has indicated that the lymphocytopenia is unrelated to the destruction of lymphocytes but is rather a consequence of an acute redistribution of recirculating lymphocytes so that they are arrested in the lymph nodes, the spleen, and especially the bone marrow at the expense of the vascular compartment (7-l 2). The evidence for this includes the reversible fall in thoracic duct output following the administration of prednisolone (9) and the increased localization of labelled lymphocytes in the bone marrow of corticosteroid-treated recipients (8). 407 0008-8749/82/020407-16$02.00/O Copyright 0 1982 by Academic Press. Inc. AU rights of reproduction in any form reserved.
408
COX AND FORD
The retention of lymphocytes within normal traffic areas of lymphoid tissue appears to be a sufficient explanation for the deficit of cells in the blood since under normal conditions recirculating lymphocytes leave the blood very rapidly to enter principally the spleen, lymph nodes, and bone marrow (13, 14). If the efflux of lymphocytes from the blood is impeded, for example by dextran sulphate administration, the blood lymphocyte level quickly rises ( 15). Corticosteroids apparently have the opposite effect but their influence on the rate at which lymphocytes leave the blood to enter various tissues has not been critically examined. This seems to deserve attention since the anti-inflammatory effect of corticosteroids has been partly attributed to a reduction in the permeability of vascular endothelium to macromolecules (16). It has also been shown that the rate of cellular exudation into inflammatory sites is diminished by corticosteroids (17). The primary aim of the present experiments was to observe the effect of prednisolone on the rate at which thoracic duct lymphocytes (TDL) leave the blood to enter the lymph nodes, spleen, bone marrow, and other organs that are important in the economy of lymphocyte recirculation. A secondary aim was to investigate further the effect of prednisolone on the traffic of lymphocytes within the tissues. MATERIALS
AND METHODS
Principle. Recipients of iv-injected radioactively labelled thoracic duct lymphocytes (TDL) from syngeneic donors were killed at various time intervals and the localization of the lymphocytes in different organs was determined by radioactive counting. The distribution of lymphocytes in recipients treated with prednisolone was compared to the distribution in untreated control recipients. Animals. Rats belonging to the highly inbred A0 strain were used in most experiments; in some experiments (A0 X DA)Fi hybrids were used. Donors of TDL were males of lo-16 weeks; recipients were females or males of 8-12 weeks. Operation. Thoracic duct cannulation was performed by Gowans’ method as described by Ford ( 18). Lymph was collected overnight at 0°C into flasks containing 100 units of heparin. Radioactive labelling and counting. Lymphocytes were labelled in vitro with L[4,5-3H]leucine (TRK.170) at 10 &i/ml, [5-‘Hluridine (TRA.178) at 20 &i/ ml, or sodium [ “Crlchromate (CJS-1P) at 10 &i/ml by the methods previously described (18). Scintillation counting and autoradiography were performed as described in the same reference (18). The distribution of labelled lymphocytes. Under light ether anaesthesia each recipient was given an injection into the lateral tail vein of 100-200 X lo6 labelled TDL in a volume of 2 ml. Recipients were killed at 0.5, 2.5, 9 and 24 hr after injection. These time intervals were chosen in order to gauge the initial distribution from the blood (0.5 hr), the maximum localization in the spleen (ca, 2.5 hr), and the distribution when the injected cells were approaching equilibrium with the rats’ own recirculating lymphocyte pool (24 hr). Before each recipient was killed a 2ml sample of blood was obtained by cardiac puncture and then the rat was killed by cervical dislocation. The other organs were removed and the radioactivity was determined per gram of organ weight and per milliliter of blood as described previously (19). In some experiments small samples of lymph nodes and spleen were fixed in cacodylate-buffered glutaraldehyde and processed by standard pro-
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
409
cedures for light or electron microscopy. Whole tibias were fractured transversely and fixed in 10% neutral-buffered formalin; 24 hr later the bone marrow plug was carefully removed and processed. This technique avoided the use of a decalcifying agent, which previously had been shown to interfere with autoradiography. Prednisolone-21-sodium succinate (Sigma) was dissolved at 0.5 mg/ml in Dulbecco’s solution A + B with 1 unit of heparin/ml (DAB-l) and continuously infused into the lateral tail vein at a rate of 2 ml/hr for 15-18 hr before the injection of labelled cells. The infusion was continued at the same rate until the rat was killed. During the infusion the rat was maintained in a Bollman restraining cage. Antigenic
Stimulation
In one series of experiments rats were given 0.1 ml of a 10% suspension of 3X washed sheep erythrocytes in PBS subcutaneously into the right footpad in order to stimulate a local immune response in the draining popliteal lymph node (20). The left feet were not injected. Three days later the rats were infused with prednisolone and injected with “0-1abelled TDL as described above. Measurement
of Blood Flow
The S6RbC1method of Sapirstein (21) was applied precisely as previously described (20). In brief, under ether anaesthesia 100 &i of 86RbC1in 0.5 ml PBS was injected iv 45 set before an injection of 0.5 ml saturated KC1 to stop the heart. Blood flow and lymphocyte influx were measured in the same lymph nodes by adjusting the windows of an LKB gamma counter to count 86Rb and “Cr simultaneously. The 86Rb activity found in lymph nodes was expressed relative to the S6Rbactivity in the kidneys to give an index of relative blood flow. Presentation
of Results
In order to interpret the distribution of lymphocytes in various organs the results were calculated as the percentage of injected activity per organ and also per gram of tissue although the results are only expressed in the latter form. Table 2 gives the weights of lymph nodes and some other organs in treated and control groups. RESULTS (I) The Distribution of Lymphocytes Corticotropin, or Metyrapone
After a Single Large Dose of Prednisolone,
In the first series of experiments a single intraperitoneal injection of prednisolone (1.25 or 5 mg) given 10 min before an iv injection of labelled lymphocytes failed to significantly alter the localization of lymphocytes except for an increase in the bone marrow localization between 2.5 and 6 hr after injection; this difference had disappeared by 24 hr. At 6 hr after the injection of prednisolone the mean thymus weight was 60% of that of the controls. The injection of corticotropin (“Synacthen,” Ciba-Geigy) at a dose sufficient to stimulate endogenous corticosterone release (40 pg/kg), 10 min before the injection of labelled lymphocytes, also caused an increase in the bone marrow localization at 2.5 hr after injection, but no other significant changes were observed. The injection of 30 mg/kg of metyrapone
410
COX AND FORD
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FIG. 1. Localization of S’Cr-labelled lymphocytes in prednisolone-treated (0) and control (0) recipients 0.5 (n = 5) 2.5 (n = 7) 9 (n = 7) and 24 (n = 9) hr after iv injection. The values plotted are the mean % of the injected doseper gm of tissue (except for thoracic duct lymphocytes). Standard deviations are not plotted, for clarity, but are recorded in Ref. (29). The approximate values for whole organs can be calculated by reference to Table 2 or Ref. (19). Note that the total weight of bone is approximately 10 g. Nearly 40% of the injected dose was present in the bone marrow of prednisolone-treated recipients.
(“Metopirone,” Ciba-Geigy) to inhibit endogenous corticosterone release failed to alter lymphocyte distribution at all time intervals. The organ localization values in all these groups and in the simultaneously injected control recipients were closely similar to the control group in Fig. 1 and are therefore not documented. (2) The Localization of Lymphocytes After Prolonged iv Infusion of Prednisolone Despite the failure of a single iv dose of prednisolone to influence lymphocyte migration from the blood, further experiments were done on the effect of infusing prednisolone at a rate of 1 mg/hr for 15-18 hr before injection of lymphocytes. Recipients were killed at four time intervals (0.5, 2.5, 9, and 24 hr) during which the infusion of prednisolone was continued. Control recipients were either restrained and infused with DAB-l or neither restrained nor infused. Comparison of lymphocyte localization values revealed dramatic differences between treated and control recipients but there were no differences between the two sets of control values which were therefore plotted together (Fig. 1).
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
411
As early as 30 min after iv injection the localization of lymphocytes in bone marrow was increased by prednisolone. This readily accounted for a slight decrease in the numbers in the blood at this early interval and a similar small decrease in numbers in the spleen and cervical lymph node. The number of cells in the mesenteric lymph node was decreased much more and this could not be wholly accounted for by the small deficit in the blood. As expected from previous studies (19) the number of lymphocytes in the blood of control recipients reached a nadir at 2.5 hr after injection and later showed a substantial recovery. In the treated recipients the blood level at 2.5 hr was much lower and there was no recovery at all, so that at 24 hr the blood level was less than 5% of that of the controls. At 2.5 hr after injection the spleens of the treated rats had received slightly fewer lymphocytes than those of control rats but the substantial fall in the splenic localization value between 2.5 and 24 hr, which reflects the release of lymphocytes from the spleen (19), failed to occur in the treated rats, so that by 24 hr there were more lymphocytes in the spleen of the treated rats. The numbers of lymphocytes in the lymph nodes and bone marrow of the treated rats stayed nearly constant between 2.5 and 24 hr, in great contrast to the controls (Fig. 1). The localizaton of lymphocytes in liver, lung, small intestine, and thymus is given in Table 1. Where there was a substantial difference between treated and controls it could be accounted for by the lower blood levels in the former. (3) The Effect of Prednisolone to Lymph
on the Recirculation
of Lymphocytes from Blood
The cumulative recovery of cell-associated radioactivity from the thoracic duct lymphocytes of recipients which were cannulated and then injected iv with “Crlabelled TDL is shown in Fig. 1. Lymph was collected from five prednisolonetreated and seven control recipients in 90-min fractions using an LKB fraction collector. In this case the difference between the treated and controls diverged even more than was the case in any of the tissues. Over the 24-hr period the recovery of cellassociated radioactivity was less than 1% of the injected dose, compared to 15% in the controls. The pattern of recovery in the prednisolone-treated recipients failed to show the 15- to 1%hr peak usually found in untreated recipients. (4) The Effect of Prednisolone Blood Flow
on the Weights of Lymphoid
Organs and their
In order to interpret the effect of prednisolone on the localization of lymphocytes in tissues it was essential to obtain information on how these two quantities were affected by prednisolone. The sequence of changes in weight of lymphoid organs is presented in Table 2. After 15-18 hr the weights of all the lymphoid organs had fallen considerably, especially the lymph nodes, which reached about half the weights of the controls. The weight loss continued throughout the period of the prednisolone infusion. During this period there was no significant change in weight of the lymphoid organs of the control recipients. Since changes in blood flow may influence the distribution of lymphocytes between different organs the blood flow was measured 1 hr after the injection of
0.71 + 0.31
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Small intestine
3
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0.1
Control
on Lymphocyte
0.82 r 0.18
1.35 + 0.8
0.85 + 0.45
0.05 + 0.01
Prednisoloneb
2.5 hr”
Nofe. See Fig. 1 for localization in other compartments. ’ Time after injection of labelled lymphocytes. ’ Prednisolone infusion for 15- 18 hr before injecting labelled TDL. * Difference between adjacent values significant, P i 0.01. *** Difference between adjacent values significant, 0.05 > P > 0.02.
+ 2.2
0.09 + 0.05
Thymus
Prednisolone*
0.5 hr”
Infusion
1 Localization
in Four Compartments
+ 0.15
0.75 ?I 0.12
1.33 + 0.3
1.28 + 0.7
0.2
Control
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0.32 + 0.12
0.65 + 0.2
0.05 f 0.04
Prednisoloneb
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1.1 + 0.56
1.66 + 0.63
0.99 + 0.32
0.2
Control
Percentage injected dose per gram (mean k SD from 5-9 rats)
The Effect of Prednisolone
TABLE
+ 0.29
0.99 + 0.56
0.24 + 0.08
0.6
0.09 + 0.09
Prednisoloneb
*
24 hf
0.89 + 0.49
1.94 + 0.86
0.88 f 0.44
0.16 z!z0.11
Control
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=
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120+
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Control
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Prednisolone
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*
*
z
303 k 90
123 k 23
163 k 42
440 + 55
Control
5 13 19
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*
:
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320 + 101
;
Control
Prednisolone
Control
40 (24)
Prednisolone
25 (9)
Organs
18.5 (2.5)
’ Time after iv injection of “Cr-1abelled TDL. *Weight in mg + SD from 4-8 rats. * Difference between adjacent values significant, P -c 0.01. ** Difference between adjacent values significant, 0.02 > P > 0.01. *** Difference between adjacent values significant, 0.05 > P > 0.02.
Thymusb
61rt25
Mesenteric
LNb
63-cll
244 f 34
Cervical LN*
Spleenb
Prednisolone
16 (0.5)
on the Weight of Lymphoid infusion
Infusion
2
Time (hr) after start of prednisolone
The Effect of Prednisolone
TABLE
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414
COX AND FORD TABLE 3 The Effect of Prednisolone Infusion on the Blood Flow Through Major Lymphoid Organs
Blood flow per gram of tissue’ Prednisolone’ z*
Percentage of injected dose per gramb
ControP
Prednisolone’
Controld
6.0 + 0.72
46.5 + 9.4
53.3 + 2.9 7.5 + 3.5
Spleen
4.9 2 0.73
Cervical LN
5.7 f 1.3
5.8 + 0.9
5.2 + 1.8
Mesenteric LN
6.2 k 2.3
5.0 f 0.6
3.3 f 1.5
:
7.2 f 2.3
Bone
1.5 f 0.3
1.4 z!z0.23
3.6 + 0.6
*
1.5 It 0.37
Note. Values represent mean f SD. ’ Blood flow-*6Rb activity in the tissue as % of total s6Rbactivity in both kidneys and then expressed per gram of tissue. b 5’Cr-labelled TDL injected iv 60 min previously. ’ Prednisolone infusion 12- 15 hr before injecting labelled TDL, seven animals. d DAB-l infusion 12-15 hr before injecting labelled TDL, four animals. * Difference between values significant, P < 0.01. ** Difference between values significant, 0.02 z P > 0.01. *** Difference between adjacent values significant, 0.05 > P > 0.02.
5’Cr-labelled lymphocytes in prednisolone-infused rats or in controls. The results presented in Table 3 show no significant difference in the blood flow per gram of bone marrow or lymph node, but a slight reduction in splenic blood flow in the treated rats. The blood flow to other major organs was unchanged. At 1 hr the lymphocyte localization was reduced in lymph nodes and increased in the bone marrow of the treated rats as expected. (5) Quantitative Autoradiographic Studies of Lymphocytes and Lymph Nodes of Prednisolone-Treated Rats
in the Bone Marrow
The measurements of “Cr activity in whole organs cannot answer several pertinent questions, including: (i) is the excess of cells in the bone marrow of prednisolone-treated rats predominantly outside the blood vesselsor is it retained within venous sinuses? and (ii) does prednisolone alter the time taken for lymphocytes to cross HEVs while entering lymph nodes? Because “Cr is an unsuitable isotope for tissue autoradiography these experiments were performed with [ 3H]leucine-labelled lymphocytes. The bone marrow and mesenteric and cervical lymph nodes were removed from prednisolone-treated and control rats at 10 and 60 min after iv injection of [ 3H]leucine-labelled TDL. Table 4 represents the distribution of labelled lymphocytes between the extravascular and intravascular compartments of bone marrow. At 10 min after injection a greater proportion of lymphocytes had crossed the blood vessel walls in the prednisolone-treated rats and by 60 min the great majority of cells in both groups were extravascular. These results are clearly not compatible with the notion that prednisolone simply holds lymphocytes within the vascular sinuses of bone marrow. The localization of lymphocytes in the bone marrow of prednisolone-treated rats
PREDNISOLONE
ARRESTS
LYMPHOCYTE TABLE
415
RECIRCULATION
4
Labelled Lymphocytes in Bone Marrow: Distribution between Intravascular and Extravascular Compartments Percentage of injected radioactivity in marrow of one tibia
Percentage of labelled cells outside blood vessels“
Time after iv injection (min)
Prednisolone
Control
Prednisolone
Control
10 60
II
96
66 99
6.2 7.4
2.1 5.4
’ &3000 cells scored for each value.
was more than double that in control rats as early as 10 min after injection. This strongly suggests that the blood vessels in bone marrow were more efficient in capturing lymphocytes from the blood since selective retention of the normal lymphocyte traffic to the bone marrow could not have produced an excess of cells so soon. The location of lymphocytes in lymph nodes in relation to HEVs is represented graphically in Fig. 2. In both the cervical and mesenteric LN lymphocytes took a longer time to cross HEVs in prednisolone-treated rats. The difference was particularly striking at 60 min when 98-99’S of the injected lymphocytes had crossed HEVs to reach the extravascular compartment of the control rats, but about 50% 100 c Cervical
b 10
Lymph
Node
1 60 Minutes
Mesenferic
Lymph
Node
‘9 ä
1
10
60
after i.v. injection
FIG. 2. Autoradiographic estimates of the rate at which labelled lymphocytes cross HEVs in LN of prednisolone-treated (0) control (@) recipients. The numerator of the % is the number of labelled cells either in the lumen or in the wall of HEVs. The denominator is all the labelled cells in the LN. The shaded area is the fraction of labelled cells in the lumen of HEV. In both groups almost all the labelled cells not associated with HEV had already crossed into the parenchyma of the LN. The number of cells that had crossed the HEV by 60 min in the treated recipients was equal to or less than the number that had crossed by 10 min in the control recipients.
416
COX AND TABLE The Effect of Antigenic
Stimulation
FORD 5
and Prednisolone on Lymph Nodes
Percentage of injected dose per gram”
Lymph node Right popliteal LN (sheep RBCs)
Prednisoloneb
Weight (mg) Prednisoloneb
Control
13.4 + 2.1 *
8.7 + 3.0
*
9.2 f
* Left popliteal LN (unstimulated)
Mesenteric
LN
14.1 +
3.0
9.9 f 5.4
3.8 f
3.9
1.01
1.7
0.26
* 1.0
5.1 f
:
10.4 f 2.9
89
f 36
* 138
+42
0.64
5.6 f 2.3 * 10.3 f 3.9
97
f 35
:139
+43
0.38
10.2 k 2.3
Cervical LN
Control
*
3.4 f 1.3 :
Relative influx in prednisolonetreated LN’
Note. Values represent mean k SD; n = 10. a “Cr-labelled TDL injected iv 60 min previously. ’ Prednisolone infusion for 12 hr before injecting labelled TDL. ’ % of injected dose in LN of prednisolone-treated rats/% of injected dose in LN of control. * Difference between adjacent values significant, P < 0.01. ** Difference between adjacent values significant, 0.02 > P > 0.01.
were still associated with HEVs in the prednisolone-treated rats. It is notable that of that 50% very few were still in the lumen of the vessels; most were within the vessel walls, suggesting that they may have been held up while in the endothelium before penetrating the basement membrane. (6) Antigenic Stimulation on Lymphocyte Influx
of a Lymph Node Modijies
the Effect of Prednisolone
Figure 1 indicates that the inhibition of lymphocyte influx following prednisolone treatment varied between different lymph nodes. In order to determine whether this could be related to differences in the level of stimulation by environmental antigens, rats were injected with 0.10 ml of 10% sheep RBCs into their right footpads 3 days before starting prednisolone infusion and injecting labelled lymphocytes iv. Table 5 shows that the antigenic stimulation increased the lymph node (LN) weight by a factor of 3 and the lymphocyte influx in exact proportion so that the lymphocyte influx per gram did not change. This is in complete accord with a previous study of the effect of this antigen on lymphocyte influx to the popliteal LN (20). The data on the prednisolone-treated LN point to a clear-cut conclusion, which is that antigenic stimulation completely protects LN from the reduced influx consequent upon prednisolone administration. Thus, in contrast to all other LN, the stimulated popliteal LN received a greater lymphocyte influx per gram in the prednisolone-treated recipients. Since prednisolone reduced the weights of both stimulated and unstimulated LN, another way of looking at these results is that while the lymphocyte influx into antigenically stimulated LN is precisely the same
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
417
in treated and control groups, the influx into nonstimulated popliteal LN is reduced by prednisolone by a factor of 4 (Table 5, final column). This effect of prednisolone is greatest in the unstimulated popliteal LN, which are particularly quiescent as judged by lack of germinal centres, and least in the cervical LN, which appear to be heavily stimulated in A0 rats as testified by an exceptional increase in weight between 3 and 6 weeks of age (unpublished observations). (7) Histological Nodes
and Ultrastructural
Observations on Prednisolone-Treated
Lymph
As expected, prolonged administration of prednisolone led to the dissolution of germinal centres with many pyknotic cells in the follicular areas. There was a reduced number of small lymphocytes in both the paracortex and the superficial cortex. On the other hand the lymph node medulla appeared to be unaffected. The height of the endothelial cells of HEVs from lumen to basement membrane was measured using an eyepiece graticule. No significant difference was found between the prednisolone-treated and control rats. Electron microscopic observation of HEVs did not suggest that prednisolone had brought about any change in the ultrastructure of any of the organelles of these cells. However, one interesting observation was that there appeared to be a definite increase in the number of darkly staining granules in the HEV cells of treated rats. This was also observed in light microscope sections; the granules were periodic acid-Schiff positive and stained very heavily with methenamine silver. There was an obvious deficiency of small lymphocytes associated with HEVs, no doubt as a consequenceof the paucity of cells in the blood. (8) Supplementary nisolone-Treated
Studies of the Viability Rats
of Lymphocytes
Injected into Pred-
In order to determine whether cell death was to any extent responsible for the differences in localization of the injected lymphocytes between prednisolone-treated and control recipients, an audit of radioactivity in all the tissues detailed in Fig. 1 and Table 1 was drawn up. For some organs such as the spleen, thymus, and mesenteric LN the total radioactivity was measured directly because the whole organ was inserted into a vial for gamma counting. For other organs such as the liver, lung, gut, and bone, where samples of the tissue were removed, total radioactivity was estimated by multiplying the mean percentage injected radioactivity per gram of sample by the mean organ weights of a “standard” 200-g rat (19). The results showed clearly that there was no significant difference between the two groups; at all time intervals the total recovery from both prednisolone and untreated groups was between 70 and 80% of the injected dose. At 24 hr after the injection of labelled TDL, the low LN recovery was fully compensated by the higher bone marrow and spleen recoveries. In particular the liver localization was lower in the treated than untreated rats. The conclusion that cell death was not responsible to any extent for the differences in distribution was fortified by two experiments. First, labelled cells from the bone marrow of treated and untreated primary recipients were retransferred into secondary recipients. Their distribution patterns were almost identical at 0.5,2.5, and 24 hr after transfer (not documented). These results must be treated with some caution, since only small numbers of
418
COX AND FORD 5 4
Bone (tibia)
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Time after stopping Prednisolone (hours) FIG. 3. Localization of S’Cr-labelled lymphocytes in prednisolone-treated (0) (n = 6-7) and control (0) (n = 4-6) recipients after stopping the administration of prednisolone as described in the text. The values plotted are the mean 8 of the injected dose per gm of tissue. Standard deviations are recorded in Ref. (29). The approximate values for whole organs can be calculated by reference to Table 2 or Ref. (19).
lymphocytes could be recovered from the bone marrow of untreated rats for injection into secondary recipients. Second, in some recipients of labelled TDL, the prednisolone infusion was stopped after 24 hr and rats were killed 3, 6, 12, and 24 hr later. The high bone marrow localization started to decrease within 3 hr, thus increasing the blood level and later the LN localization (Fig. 3). Twenty-four hours after stopping prednisolone the localization in LN was greater than in the untreated rats, but even by this time the bone marrow localization in the prednisolone-treated rats, though still falling, had not reached the level of the untreated rats (Fig. 3). (9) Differential Effect of Prednisolone on the Traffic of T and B Lymphocytes Studies in humans (5) and guinea pigs (8) have suggested that the effect of corticosteroids in arresting lymphocyte traffic is greater on T cells than on B cells.
PREDNISOLONE
ARRESTS
LYMPHOCYTE
419
RECIRCULATION
This proposition was investigated in rats by exploiting Howard’s finding that T lymphocytes take up lo- 15 times more [ 3H]uridine in vitro than do B lymphocytes (22). TDL labelled in vitro with [3H]uridine were injected iv into prednisolonetreated or control rats which were killed 1 hr later. Autoradiographs of spleen, cervical LN, and bone marrow were exposed for several periods so that a time could be chosen at which there was a clear distinction between heavily labelled cells with areas of confluent grains and lightly labelled cells without confluence. In the control recipients 36-40s of the TDL that had entered the three tissues were lightly labelled, corresponding to the expected proportion of B cells (22) (Table 6). In the prednisolone-treated rats there was little difference in any of the three tissues, indicating that the traffic of B and T lymphocytes had each been perturbed in the same direction. There was a slightly increased proportion of heavily labelled T cells in bone marrow and correspondingly a slightly decreased proportion in the LN and spleen (Table 6), suggesting that prednisolone may exert a marginally greater effect on T lymphocyte entry into the bone marrow. DISCUSSION Previous studies of the impact of corticosteroids on the immune system have concentrated on describing changes in the weight and histology of lymphoid organs (23) and changes in lymphocyte concentrations in the blood (e.g., (5, 8)) and thoracic duct lymph (9, 24) after single or multiple doses of steroid. The novelty of the present study lies in following a population of recirculating lymphocytes in syngeneic recipients receiving a continuous infusion of a large (but apparently not harmful) dose of prednisolone. It was confirmed that corticosteroids kill lymphocytes in at least two compartments-the thymus and germinal centres in peripheral lymphoid tissue. However, neither of these compartments are in rapid equilibrium with the recirculating lymphocyte pool and therefore their complete destruction would not be expected to have an immediate effect on the number of recirculating lymphocytes. The present results firmly indicate that recirculating lymphocytes are not destroyed in a prednisolone-treated recipient. First, equal numbers can be accounted for in prednisolone-treated and control recipients. Second, the distribution of reTABLE
6
The Effect of Prednisolone Infusion on T and B Lymphocytes Prednisolone”
Cervical lymph node Bone marrow Spleen
Control
% Heavily labelledb
% Lightly labelled
% Heavily labelledb
% Lightly labelled
58.0 65.2 57.3
42.0 34.8 42.1
61.0 63.5 60.2
39.0 36.5 39.8
Note. [‘H]Uridine-labelled TDL injected iv 1 hr previously; 4000-5000 compartment. a Rat given an iv infusion of prednisolone 12-15 hr before injection. * Corresponds approximately to proportion of T lymphocytes (22). ’ Corresponds approximately to proportion of B lymphocytes (22).
cells were counted in each
420
COX AND
FORD
circulating lymphocytes starts to return towards that found in the control recipients within a few hours of stopping the prednisolone. And third, the excess of cells in the bone marrow of prednisolone-treated rats survive and migrate normally after retransfer to secondary recipients. It can be concluded that the profound deficit of small lymphocytes in the blood of prednisolone-treated rats, which affects both transferred and indigenous lymphocytes, is a consequenceof interference with the physiological migration pattern of lymphocytes. The retention of recirculating lymphocytes in the normal traffic areas has been suggested as the predominant factor in the lymphocytopenia (712). The present results support this notion in that they indicate prolonged retention of recirculating lymphocytes in the bone marrow, spleen, and lymph nodes of prednisolone-treated rats. Thus the high bone marrow localization 30 min and 2.5 hr after injection did not decreaseas long as the prednisolone infusion was maintained, in contrast to the control situation, in which a large decrease in marrow localization occurred between 2.5 and 9 hr after injection indicating a rapid transit time through bone marrow as argued by Rannie and Bell (25). Similarly, in the spleen the retentive effect of prednisolone is indicated by the maintenance of a high localization between 2.5 and 24 hr in contrast to the substantial redistribution from the spleen seen in the controls. In lymph nodes the transit time of lymphocytes recirculating from blood to lymph averages about 15- 18 hr for T cells as gauged by the modal recovery time from a thoracic duct fistula. Prednisolone treatment drastically inhibits this recovery although the site within LN at which cells are delayed is not known. In conclusion, prednisolone freezes the recirculation of lymphocytes within the tissues at which they arrive from the blood. The next question to consider is whether the sole effect of prednisolone on lymphocyte recirculation is retention in the tissues. The substantial reduction of the early (0.5 hr) localization of lymphocytes in the mesenteric LN suggests the possibility that prednisolone interferes to some extent with the influx of lymphocytes into LN, presumably by interfering with the adhesion of lymphocytes to and their migration across HEVs. The localization of lymphocytes in LN is directly proportional to the mean concentration in the perfusing blood (26); thus there is a greater deficit at 0.5 hr in the mesenteric LNs (39% of untreated) than would be expected from the blood levels (72% of untreated) and the integrated deficit in the blood over the relevant period (O-O.5 hr) was no doubt between 72 and 100% because the blood content of prednisolone and control recipients each started as 100% of the injected dose at time zero and after 0.5 hr continued to diverge. Clinching evidence for an inhibitory effect on lymphocyte influx came from the experiments on the popliteal LNs. Prednisolone treatment reduced the influx into the quiescent left popliteal LN to 26% of the untreated control, but the antigenically stimulated right popliteal LN showed no deficit at all (101% of the control, Table 5). Apparently the lymphocyte influx into the unstimulated lymph node is substantially inhibited by prednisolone, but antigenic stimulation 3 days previously exempts a LN from this inhibition. The significance of this exemption may be that it allows a stimulated LN to continue to recruit antigen-specific lymphocytes from the recirculating pool in the face of high levels of endogenous corticosteroids. This striking finding should be taken further by investigating the time relationship between the injection of antigen and the exemption of the LN from the inhibitory effect of prednisolone on lymphocyte influx. Two groups have found that the immunosuppressive effect of corticosteroids is most marked when given l-3 days
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
421
before antigenic stimulation; there is little or no suppressive effect when they are administered l-3 days after antigen (27-28). We suggest that established immune responsesmay be invulnerable to corticosteroid suppression because of the exemption of stimulated LN from inhibition of lymphocyte influx. Prednisolone did not produce any obvious structural change in HEVs apart from an increase in small granules staining with periodic acid Schiff. The autoradiographic analysis of the transit through the wall of HEVs suggestedthat prednisolone prolonged the time taken for lymphocytes to travel from the lumen to beyond the basement membrane. In A0 rats this process normally takes about 5- 15 min (Fig. 2 and unpublished data of W. L. Ford and M. E. Smith), but prednisolone treatment increases this time to more than 60 min. The difference is not because lymphocytes take a longer time to arrive at the LN from the blood in the prednisolone group; on the contrary the bulk of lymphocytes arrived more promptly in the treated group. Moreover, there was no significant difference in LN blood flow in the two groups. Lymphocytes passed between the endothelial cells of the HEVs in the prednisolone-treated rats but accumulated on the vascular side of the basement membrane as if unable to penetrate it (29). The bone marrow was the predominant site of the excess of lymphocytes in the prednisolone-treated group, in conformity with previous studies (8, 10, 12). The extraordinary rapidity with which this excessdeveloped (10 min after iv injection) argued that prednisolone facilitated the extraction of lymphocytes from the blood by vascualr endothelium in bone marrow. Thus prednisolone exerted opposite effects on the entry of lymphocytes into bone marrow and their entry into lymph nodes. The effects of prednisolone described in this paper may be primarily on lymphocytes themselves or primarily on other structures such as vascular endothelium, macrophages, and reticular structures. The exposure of mouse lymphocytes in vitro to hydrocortisone at 0.2 mg/ml for 4 hr did not affect their distribution in vivo after iv injection, but, not surprisingly, 2 mg/ml for 4 hr did impair their migration (30). In a thorough study more physiological concentrations of hydrocortisone have been found to inhibit the chemotaxis of rat lymphocytes in vitro with a gradation of effect from lo-’ to 10m3M (3 1). Our findings that (a) a single large dose of prednisolone had little effect on lymphocyte distribution, and (b) lymphocytes transferred from a prednisolone-infused rat to an untreated secondary recipient migrated normally, are both most easily explained by an indirect or secondary effect on lymphocytes, but other explanations are certainly possible. The traffic of lymphocytes is vitally important to both the initiation and development of immune responses(14, 32). The arrest of lymphocyte traffic as described here may contribute substantially to the immunosuppressive effects of corticosteroids, but its importance relative to the other possible mechanisms is difficult to judge at present. ACKNOWLEDGMENTS We wish to thank Mrs. N. Rattray and Mrs. T. Aslan for technical assistance.The work was largely supported by MRC Programme Grant G 977/455/B. J.H.C. held an MRC research studentship.
REFERENCES 1. Baxter, J. D., and Harris, A. W., Transplant. Proc. 7, 55, 1975. 2. Fauci, A. S., In “Monographs in Endocrinology,” Vol. 12, “Glucocorticoid Hormone Actions” (J. D. Baxter and G. G. Rousseau, Eds.), Chap. 24. Springer-Verlag, Berlin, 1979.
422
COX AND
FORD
3. Dougherty, T. F., Physiol. Rev. 32, 319, 1952. 4. Dougherty, T. F., Berliner, M. L., and Schneebeli, G. L., Ann. N. Y. Acad. Sci. 113, 825, 1964. 5. Fauci, A. S., and Dale, D. S., J. Clin. Invest. 53, 240, 1974. 6. Van Dijk, H., Bloksma, N., Rademaker, P. M., Schouten, W. J., and Willers, J. W., Int. J. Immunopharmacol.
1, 285, 1979.
7. Lundin, P. M., and Hedman, L. A., Lymphology 11, 216, 1978. 8. Fauci, A. S., Immunology 28, 669, 1975. 9. Spry, C. J. F., Cell. Immunol. 4, 86, 1972. 10. Cohen, J. J., J. Immunol. 108, 841, 1972. 11. Fauci, A. S., Transplant. Proc. 7, 37, 1975. 12. Moorhead, J. W., and Claman, H. N., Cell. Immunol. 5, 74, 1972. 13. Gowans, J. L., and Knight, E. J., Proc. R. Sot. London Ser. B 159, 257, 1964. 14. Ford, W. L., Progr. Allergy 19, 1, 1975. 15. Ford, W. L., Smith, M. E., and Andrews, P., In “Cell-Cell Recognition, Society for Experimental Biology Symposium No. 32” (A. S. G. Curtis, Ed.), pp. 359-392. Cambridge Univ. Press,London/ New York, 1978. 16. Cope, C. L., “Adrenal Steroids and Disease,” 2nd ed. Pitman, London, 1972. 17. Perper, R. J., Sanoa, M., Chinea, G., and Oronsky, A. L., J. Lab. C&z. Med. 84, 394, 1974. 18. Ford, W. L., In “Handbook of Experimental Immunology” (D. M. Weir, Ed.), 3rd ed., Chap. 23. Blackwell, Oxford, 1978. 19. Smith, M. E., Martin, A. F., and Ford, W. L., Monogr. Allergy 16, 203, 1980. 20. Drayson, M. T., Smith, M. E., and Ford, W. L., Immunology 44, 125, 1981. 21. Sapirstein, L. A., J. Appl. Physiol. 193, 161, 1958. 22. Howard, J. C., Hunt, S. V., and Gowans, J. L., J. Exp. Med. 135, 200, 1972. 23. Dracott, B. N., and Smith, C. E. T., Immunology 38, 429, 1979. 24. Schnappauf, H., and Schnappauf, U., Nouv. Rev. Fr. Hematol. 8, 555, 1968. 25. Rannie, G. H., and Bell, E. B., Trunsplantation 27, 369, 1979. 26. Sedgley, M., and Ford, W. L., Cell Tissue Kinet. 9, 231, 1976. 27. Dracott, B. N., and Smith, C. E. T., Immunology 38, 437, 1979. 28. Elliott, E. V., and Sinclair, N. R. St C., Immunology 15, 643, 1968. 29. Cox, J., Ph. D. thesis, University of Manchester, 1981. 30. Lance, E. M., and Cooper, S., In “Ciba Foundation Study Group No. 36” (G. E. W. Wolstenholme and J. Knight, Eds.), 73, 1970. 31. Beer, D. J., and Center, D. M., Cell. Immunol. 55, 381, 1980. 32. Gowans, J. L., and McGregor, D. D., Progr. Allergy 9, 1, 1965.
IMMUNOLOGY
66,407-422
The Migration
IV. Prednisolone
(1982)
of Lymphocytes across Specialized Vascular Endothelium
Acts at Several Points on the Recirculation of Lymphocytes JOSEPHINE
Pathways
H. Cox AND W. L. FORD
Experimental Pathology Laboratory, Department of Pathology, Stopford Building, University of Manchester. Manchester MI 3 9PT, United Kingdom Received November 4, 1981: accepted November 23, 1981 Radioactively labelled thoracic duct lymphocytes from syngeneic rat donors were injected iv into recipients which had been given a continuous iv infusion of prednisolone at 1 mg/hr for 15-18 hr previously. The tissue distribution and recirculation into lymph of the labelled lymphocytes were compared quantitatively in the prednisolone-treated and control recipients by scintillation counting and autoradiography. The most prominent effect of prednisolone was to retard recirculating lymphocytes within the tissues to which they are normally distributed by the blood, namely the bone marrow, the spleen, and the lymph nodes.Although lymphocyte traffic was almost completely frozen by prednisolone, recirculating lymphocytes were not killed. A secondeffect of prednisolone was to impair the influx of lymphocytes from the blood into lymph nodes. Different groups of lymph nodes varied in the extent to which prednisolone inhibited the entry of lymphocytes, and previous antigenic stimulation completely exempted lymph nodes from this inhibition. Lymphocytes took a longer time to cross the walls of high endothelial venules in the lymph nodes of prednisolone-treated rats. A third effect of prednisolone was to increase the rate at which lymphocytes entered the bone marrow from the blood by crossing sinusoidal endothelium.
INTRODUCTION A great deal of work over the past three decades has established that the administration of corticosteroids may produce (i) suppression of immune responses ( 1, 2); (ii) destruction of large numbers of lymphocytes particularly in the thymus and germinal centres (3, 4), and (iii) a deficit of lymphocytes in the blood, as well as many other effects. Lymphocytopenia following large doses of corticosteroids has been described in man (5), mice (6), rats (7), and guinea pigs (8). Recent work has indicated that the lymphocytopenia is unrelated to the destruction of lymphocytes but is rather a consequence of an acute redistribution of recirculating lymphocytes so that they are arrested in the lymph nodes, the spleen, and especially the bone marrow at the expense of the vascular compartment (7-l 2). The evidence for this includes the reversible fall in thoracic duct output following the administration of prednisolone (9) and the increased localization of labelled lymphocytes in the bone marrow of corticosteroid-treated recipients (8). 407 0008-8749/82/020407-16$02.00/O Copyright 0 1982 by Academic Press. Inc. AU rights of reproduction in any form reserved.
408
COX AND FORD
The retention of lymphocytes within normal traffic areas of lymphoid tissue appears to be a sufficient explanation for the deficit of cells in the blood since under normal conditions recirculating lymphocytes leave the blood very rapidly to enter principally the spleen, lymph nodes, and bone marrow (13, 14). If the efflux of lymphocytes from the blood is impeded, for example by dextran sulphate administration, the blood lymphocyte level quickly rises ( 15). Corticosteroids apparently have the opposite effect but their influence on the rate at which lymphocytes leave the blood to enter various tissues has not been critically examined. This seems to deserve attention since the anti-inflammatory effect of corticosteroids has been partly attributed to a reduction in the permeability of vascular endothelium to macromolecules (16). It has also been shown that the rate of cellular exudation into inflammatory sites is diminished by corticosteroids (17). The primary aim of the present experiments was to observe the effect of prednisolone on the rate at which thoracic duct lymphocytes (TDL) leave the blood to enter the lymph nodes, spleen, bone marrow, and other organs that are important in the economy of lymphocyte recirculation. A secondary aim was to investigate further the effect of prednisolone on the traffic of lymphocytes within the tissues. MATERIALS
AND METHODS
Principle. Recipients of iv-injected radioactively labelled thoracic duct lymphocytes (TDL) from syngeneic donors were killed at various time intervals and the localization of the lymphocytes in different organs was determined by radioactive counting. The distribution of lymphocytes in recipients treated with prednisolone was compared to the distribution in untreated control recipients. Animals. Rats belonging to the highly inbred A0 strain were used in most experiments; in some experiments (A0 X DA)Fi hybrids were used. Donors of TDL were males of lo-16 weeks; recipients were females or males of 8-12 weeks. Operation. Thoracic duct cannulation was performed by Gowans’ method as described by Ford ( 18). Lymph was collected overnight at 0°C into flasks containing 100 units of heparin. Radioactive labelling and counting. Lymphocytes were labelled in vitro with L[4,5-3H]leucine (TRK.170) at 10 &i/ml, [5-‘Hluridine (TRA.178) at 20 &i/ ml, or sodium [ “Crlchromate (CJS-1P) at 10 &i/ml by the methods previously described (18). Scintillation counting and autoradiography were performed as described in the same reference (18). The distribution of labelled lymphocytes. Under light ether anaesthesia each recipient was given an injection into the lateral tail vein of 100-200 X lo6 labelled TDL in a volume of 2 ml. Recipients were killed at 0.5, 2.5, 9 and 24 hr after injection. These time intervals were chosen in order to gauge the initial distribution from the blood (0.5 hr), the maximum localization in the spleen (ca, 2.5 hr), and the distribution when the injected cells were approaching equilibrium with the rats’ own recirculating lymphocyte pool (24 hr). Before each recipient was killed a 2ml sample of blood was obtained by cardiac puncture and then the rat was killed by cervical dislocation. The other organs were removed and the radioactivity was determined per gram of organ weight and per milliliter of blood as described previously (19). In some experiments small samples of lymph nodes and spleen were fixed in cacodylate-buffered glutaraldehyde and processed by standard pro-
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
409
cedures for light or electron microscopy. Whole tibias were fractured transversely and fixed in 10% neutral-buffered formalin; 24 hr later the bone marrow plug was carefully removed and processed. This technique avoided the use of a decalcifying agent, which previously had been shown to interfere with autoradiography. Prednisolone-21-sodium succinate (Sigma) was dissolved at 0.5 mg/ml in Dulbecco’s solution A + B with 1 unit of heparin/ml (DAB-l) and continuously infused into the lateral tail vein at a rate of 2 ml/hr for 15-18 hr before the injection of labelled cells. The infusion was continued at the same rate until the rat was killed. During the infusion the rat was maintained in a Bollman restraining cage. Antigenic
Stimulation
In one series of experiments rats were given 0.1 ml of a 10% suspension of 3X washed sheep erythrocytes in PBS subcutaneously into the right footpad in order to stimulate a local immune response in the draining popliteal lymph node (20). The left feet were not injected. Three days later the rats were infused with prednisolone and injected with “0-1abelled TDL as described above. Measurement
of Blood Flow
The S6RbC1method of Sapirstein (21) was applied precisely as previously described (20). In brief, under ether anaesthesia 100 &i of 86RbC1in 0.5 ml PBS was injected iv 45 set before an injection of 0.5 ml saturated KC1 to stop the heart. Blood flow and lymphocyte influx were measured in the same lymph nodes by adjusting the windows of an LKB gamma counter to count 86Rb and “Cr simultaneously. The 86Rb activity found in lymph nodes was expressed relative to the S6Rbactivity in the kidneys to give an index of relative blood flow. Presentation
of Results
In order to interpret the distribution of lymphocytes in various organs the results were calculated as the percentage of injected activity per organ and also per gram of tissue although the results are only expressed in the latter form. Table 2 gives the weights of lymph nodes and some other organs in treated and control groups. RESULTS (I) The Distribution of Lymphocytes Corticotropin, or Metyrapone
After a Single Large Dose of Prednisolone,
In the first series of experiments a single intraperitoneal injection of prednisolone (1.25 or 5 mg) given 10 min before an iv injection of labelled lymphocytes failed to significantly alter the localization of lymphocytes except for an increase in the bone marrow localization between 2.5 and 6 hr after injection; this difference had disappeared by 24 hr. At 6 hr after the injection of prednisolone the mean thymus weight was 60% of that of the controls. The injection of corticotropin (“Synacthen,” Ciba-Geigy) at a dose sufficient to stimulate endogenous corticosterone release (40 pg/kg), 10 min before the injection of labelled lymphocytes, also caused an increase in the bone marrow localization at 2.5 hr after injection, but no other significant changes were observed. The injection of 30 mg/kg of metyrapone
410
COX AND FORD
Blood 0.4
I P
0.2
l
.B
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l
40
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.
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60
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,
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16
20
24
ax
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1 4
8
12
16
20
24
Time after injection of cells (hours)
FIG. 1. Localization of S’Cr-labelled lymphocytes in prednisolone-treated (0) and control (0) recipients 0.5 (n = 5) 2.5 (n = 7) 9 (n = 7) and 24 (n = 9) hr after iv injection. The values plotted are the mean % of the injected doseper gm of tissue (except for thoracic duct lymphocytes). Standard deviations are not plotted, for clarity, but are recorded in Ref. (29). The approximate values for whole organs can be calculated by reference to Table 2 or Ref. (19). Note that the total weight of bone is approximately 10 g. Nearly 40% of the injected dose was present in the bone marrow of prednisolone-treated recipients.
(“Metopirone,” Ciba-Geigy) to inhibit endogenous corticosterone release failed to alter lymphocyte distribution at all time intervals. The organ localization values in all these groups and in the simultaneously injected control recipients were closely similar to the control group in Fig. 1 and are therefore not documented. (2) The Localization of Lymphocytes After Prolonged iv Infusion of Prednisolone Despite the failure of a single iv dose of prednisolone to influence lymphocyte migration from the blood, further experiments were done on the effect of infusing prednisolone at a rate of 1 mg/hr for 15-18 hr before injection of lymphocytes. Recipients were killed at four time intervals (0.5, 2.5, 9, and 24 hr) during which the infusion of prednisolone was continued. Control recipients were either restrained and infused with DAB-l or neither restrained nor infused. Comparison of lymphocyte localization values revealed dramatic differences between treated and control recipients but there were no differences between the two sets of control values which were therefore plotted together (Fig. 1).
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
411
As early as 30 min after iv injection the localization of lymphocytes in bone marrow was increased by prednisolone. This readily accounted for a slight decrease in the numbers in the blood at this early interval and a similar small decrease in numbers in the spleen and cervical lymph node. The number of cells in the mesenteric lymph node was decreased much more and this could not be wholly accounted for by the small deficit in the blood. As expected from previous studies (19) the number of lymphocytes in the blood of control recipients reached a nadir at 2.5 hr after injection and later showed a substantial recovery. In the treated recipients the blood level at 2.5 hr was much lower and there was no recovery at all, so that at 24 hr the blood level was less than 5% of that of the controls. At 2.5 hr after injection the spleens of the treated rats had received slightly fewer lymphocytes than those of control rats but the substantial fall in the splenic localization value between 2.5 and 24 hr, which reflects the release of lymphocytes from the spleen (19), failed to occur in the treated rats, so that by 24 hr there were more lymphocytes in the spleen of the treated rats. The numbers of lymphocytes in the lymph nodes and bone marrow of the treated rats stayed nearly constant between 2.5 and 24 hr, in great contrast to the controls (Fig. 1). The localizaton of lymphocytes in liver, lung, small intestine, and thymus is given in Table 1. Where there was a substantial difference between treated and controls it could be accounted for by the lower blood levels in the former. (3) The Effect of Prednisolone to Lymph
on the Recirculation
of Lymphocytes from Blood
The cumulative recovery of cell-associated radioactivity from the thoracic duct lymphocytes of recipients which were cannulated and then injected iv with “Crlabelled TDL is shown in Fig. 1. Lymph was collected from five prednisolonetreated and seven control recipients in 90-min fractions using an LKB fraction collector. In this case the difference between the treated and controls diverged even more than was the case in any of the tissues. Over the 24-hr period the recovery of cellassociated radioactivity was less than 1% of the injected dose, compared to 15% in the controls. The pattern of recovery in the prednisolone-treated recipients failed to show the 15- to 1%hr peak usually found in untreated recipients. (4) The Effect of Prednisolone Blood Flow
on the Weights of Lymphoid
Organs and their
In order to interpret the effect of prednisolone on the localization of lymphocytes in tissues it was essential to obtain information on how these two quantities were affected by prednisolone. The sequence of changes in weight of lymphoid organs is presented in Table 2. After 15-18 hr the weights of all the lymphoid organs had fallen considerably, especially the lymph nodes, which reached about half the weights of the controls. The weight loss continued throughout the period of the prednisolone infusion. During this period there was no significant change in weight of the lymphoid organs of the control recipients. Since changes in blood flow may influence the distribution of lymphocytes between different organs the blood flow was measured 1 hr after the injection of
0.71 + 0.31
14.2
0.57 k 0.11
Liver
Lung
Small intestine
3
+ 0.1
-+ 3.4
0.44 + 0.32
14.1
1.5 + 0.6
0.1
Control
on Lymphocyte
0.82 r 0.18
1.35 + 0.8
0.85 + 0.45
0.05 + 0.01
Prednisoloneb
2.5 hr”
Nofe. See Fig. 1 for localization in other compartments. ’ Time after injection of labelled lymphocytes. ’ Prednisolone infusion for 15- 18 hr before injecting labelled TDL. * Difference between adjacent values significant, P i 0.01. *** Difference between adjacent values significant, 0.05 > P > 0.02.
+ 2.2
0.09 + 0.05
Thymus
Prednisolone*
0.5 hr”
Infusion
1 Localization
in Four Compartments
+ 0.15
0.75 ?I 0.12
1.33 + 0.3
1.28 + 0.7
0.2
Control
0.9
+ 0.48
0.32 + 0.12
0.65 + 0.2
0.05 f 0.04
Prednisoloneb
*
;
9 hP
+ 0.16
1.1 + 0.56
1.66 + 0.63
0.99 + 0.32
0.2
Control
Percentage injected dose per gram (mean k SD from 5-9 rats)
The Effect of Prednisolone
TABLE
+ 0.29
0.99 + 0.56
0.24 + 0.08
0.6
0.09 + 0.09
Prednisoloneb
*
24 hf
0.89 + 0.49
1.94 + 0.86
0.88 f 0.44
0.16 z!z0.11
Control
g
z
i2 u
200 + 75
;
=
*
49
37
34
287 -+ 124
116t
120+
385 +
Control
17
193 + 48
70f
68k16
327 + 50
Prednisolone
:
*
*
z
303 k 90
123 k 23
163 k 42
440 + 55
Control
5 13 19
52+ 93* 191 +
*
:
*
35
49
185 + 384 +
42
151 f
* * *
44+- 12 41 + 21 78 + 36
338 + 90
158 + 43
164 f 45
515 + 88
*
251 t65
517 + 110
320 + 101
;
Control
Prednisolone
Control
40 (24)
Prednisolone
25 (9)
Organs
18.5 (2.5)
’ Time after iv injection of “Cr-1abelled TDL. *Weight in mg + SD from 4-8 rats. * Difference between adjacent values significant, P -c 0.01. ** Difference between adjacent values significant, 0.02 > P > 0.01. *** Difference between adjacent values significant, 0.05 > P > 0.02.
Thymusb
61rt25
Mesenteric
LNb
63-cll
244 f 34
Cervical LN*
Spleenb
Prednisolone
16 (0.5)
on the Weight of Lymphoid infusion
Infusion
2
Time (hr) after start of prednisolone
The Effect of Prednisolone
TABLE
G
5
ifi 9
2
F:
2 z
3
% cz
m
2
g
g
z
414
COX AND FORD TABLE 3 The Effect of Prednisolone Infusion on the Blood Flow Through Major Lymphoid Organs
Blood flow per gram of tissue’ Prednisolone’ z*
Percentage of injected dose per gramb
ControP
Prednisolone’
Controld
6.0 + 0.72
46.5 + 9.4
53.3 + 2.9 7.5 + 3.5
Spleen
4.9 2 0.73
Cervical LN
5.7 f 1.3
5.8 + 0.9
5.2 + 1.8
Mesenteric LN
6.2 k 2.3
5.0 f 0.6
3.3 f 1.5
:
7.2 f 2.3
Bone
1.5 f 0.3
1.4 z!z0.23
3.6 + 0.6
*
1.5 It 0.37
Note. Values represent mean f SD. ’ Blood flow-*6Rb activity in the tissue as % of total s6Rbactivity in both kidneys and then expressed per gram of tissue. b 5’Cr-labelled TDL injected iv 60 min previously. ’ Prednisolone infusion 12- 15 hr before injecting labelled TDL, seven animals. d DAB-l infusion 12-15 hr before injecting labelled TDL, four animals. * Difference between values significant, P < 0.01. ** Difference between values significant, 0.02 z P > 0.01. *** Difference between adjacent values significant, 0.05 > P > 0.02.
5’Cr-labelled lymphocytes in prednisolone-infused rats or in controls. The results presented in Table 3 show no significant difference in the blood flow per gram of bone marrow or lymph node, but a slight reduction in splenic blood flow in the treated rats. The blood flow to other major organs was unchanged. At 1 hr the lymphocyte localization was reduced in lymph nodes and increased in the bone marrow of the treated rats as expected. (5) Quantitative Autoradiographic Studies of Lymphocytes and Lymph Nodes of Prednisolone-Treated Rats
in the Bone Marrow
The measurements of “Cr activity in whole organs cannot answer several pertinent questions, including: (i) is the excess of cells in the bone marrow of prednisolone-treated rats predominantly outside the blood vesselsor is it retained within venous sinuses? and (ii) does prednisolone alter the time taken for lymphocytes to cross HEVs while entering lymph nodes? Because “Cr is an unsuitable isotope for tissue autoradiography these experiments were performed with [ 3H]leucine-labelled lymphocytes. The bone marrow and mesenteric and cervical lymph nodes were removed from prednisolone-treated and control rats at 10 and 60 min after iv injection of [ 3H]leucine-labelled TDL. Table 4 represents the distribution of labelled lymphocytes between the extravascular and intravascular compartments of bone marrow. At 10 min after injection a greater proportion of lymphocytes had crossed the blood vessel walls in the prednisolone-treated rats and by 60 min the great majority of cells in both groups were extravascular. These results are clearly not compatible with the notion that prednisolone simply holds lymphocytes within the vascular sinuses of bone marrow. The localization of lymphocytes in the bone marrow of prednisolone-treated rats
PREDNISOLONE
ARRESTS
LYMPHOCYTE TABLE
415
RECIRCULATION
4
Labelled Lymphocytes in Bone Marrow: Distribution between Intravascular and Extravascular Compartments Percentage of injected radioactivity in marrow of one tibia
Percentage of labelled cells outside blood vessels“
Time after iv injection (min)
Prednisolone
Control
Prednisolone
Control
10 60
II
96
66 99
6.2 7.4
2.1 5.4
’ &3000 cells scored for each value.
was more than double that in control rats as early as 10 min after injection. This strongly suggests that the blood vessels in bone marrow were more efficient in capturing lymphocytes from the blood since selective retention of the normal lymphocyte traffic to the bone marrow could not have produced an excess of cells so soon. The location of lymphocytes in lymph nodes in relation to HEVs is represented graphically in Fig. 2. In both the cervical and mesenteric LN lymphocytes took a longer time to cross HEVs in prednisolone-treated rats. The difference was particularly striking at 60 min when 98-99’S of the injected lymphocytes had crossed HEVs to reach the extravascular compartment of the control rats, but about 50% 100 c Cervical
b 10
Lymph
Node
1 60 Minutes
Mesenferic
Lymph
Node
‘9 ä
1
10
60
after i.v. injection
FIG. 2. Autoradiographic estimates of the rate at which labelled lymphocytes cross HEVs in LN of prednisolone-treated (0) control (@) recipients. The numerator of the % is the number of labelled cells either in the lumen or in the wall of HEVs. The denominator is all the labelled cells in the LN. The shaded area is the fraction of labelled cells in the lumen of HEV. In both groups almost all the labelled cells not associated with HEV had already crossed into the parenchyma of the LN. The number of cells that had crossed the HEV by 60 min in the treated recipients was equal to or less than the number that had crossed by 10 min in the control recipients.
416
COX AND TABLE The Effect of Antigenic
Stimulation
FORD 5
and Prednisolone on Lymph Nodes
Percentage of injected dose per gram”
Lymph node Right popliteal LN (sheep RBCs)
Prednisoloneb
Weight (mg) Prednisoloneb
Control
13.4 + 2.1 *
8.7 + 3.0
*
9.2 f
* Left popliteal LN (unstimulated)
Mesenteric
LN
14.1 +
3.0
9.9 f 5.4
3.8 f
3.9
1.01
1.7
0.26
* 1.0
5.1 f
:
10.4 f 2.9
89
f 36
* 138
+42
0.64
5.6 f 2.3 * 10.3 f 3.9
97
f 35
:139
+43
0.38
10.2 k 2.3
Cervical LN
Control
*
3.4 f 1.3 :
Relative influx in prednisolonetreated LN’
Note. Values represent mean k SD; n = 10. a “Cr-labelled TDL injected iv 60 min previously. ’ Prednisolone infusion for 12 hr before injecting labelled TDL. ’ % of injected dose in LN of prednisolone-treated rats/% of injected dose in LN of control. * Difference between adjacent values significant, P < 0.01. ** Difference between adjacent values significant, 0.02 > P > 0.01.
were still associated with HEVs in the prednisolone-treated rats. It is notable that of that 50% very few were still in the lumen of the vessels; most were within the vessel walls, suggesting that they may have been held up while in the endothelium before penetrating the basement membrane. (6) Antigenic Stimulation on Lymphocyte Influx
of a Lymph Node Modijies
the Effect of Prednisolone
Figure 1 indicates that the inhibition of lymphocyte influx following prednisolone treatment varied between different lymph nodes. In order to determine whether this could be related to differences in the level of stimulation by environmental antigens, rats were injected with 0.10 ml of 10% sheep RBCs into their right footpads 3 days before starting prednisolone infusion and injecting labelled lymphocytes iv. Table 5 shows that the antigenic stimulation increased the lymph node (LN) weight by a factor of 3 and the lymphocyte influx in exact proportion so that the lymphocyte influx per gram did not change. This is in complete accord with a previous study of the effect of this antigen on lymphocyte influx to the popliteal LN (20). The data on the prednisolone-treated LN point to a clear-cut conclusion, which is that antigenic stimulation completely protects LN from the reduced influx consequent upon prednisolone administration. Thus, in contrast to all other LN, the stimulated popliteal LN received a greater lymphocyte influx per gram in the prednisolone-treated recipients. Since prednisolone reduced the weights of both stimulated and unstimulated LN, another way of looking at these results is that while the lymphocyte influx into antigenically stimulated LN is precisely the same
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
417
in treated and control groups, the influx into nonstimulated popliteal LN is reduced by prednisolone by a factor of 4 (Table 5, final column). This effect of prednisolone is greatest in the unstimulated popliteal LN, which are particularly quiescent as judged by lack of germinal centres, and least in the cervical LN, which appear to be heavily stimulated in A0 rats as testified by an exceptional increase in weight between 3 and 6 weeks of age (unpublished observations). (7) Histological Nodes
and Ultrastructural
Observations on Prednisolone-Treated
Lymph
As expected, prolonged administration of prednisolone led to the dissolution of germinal centres with many pyknotic cells in the follicular areas. There was a reduced number of small lymphocytes in both the paracortex and the superficial cortex. On the other hand the lymph node medulla appeared to be unaffected. The height of the endothelial cells of HEVs from lumen to basement membrane was measured using an eyepiece graticule. No significant difference was found between the prednisolone-treated and control rats. Electron microscopic observation of HEVs did not suggest that prednisolone had brought about any change in the ultrastructure of any of the organelles of these cells. However, one interesting observation was that there appeared to be a definite increase in the number of darkly staining granules in the HEV cells of treated rats. This was also observed in light microscope sections; the granules were periodic acid-Schiff positive and stained very heavily with methenamine silver. There was an obvious deficiency of small lymphocytes associated with HEVs, no doubt as a consequenceof the paucity of cells in the blood. (8) Supplementary nisolone-Treated
Studies of the Viability Rats
of Lymphocytes
Injected into Pred-
In order to determine whether cell death was to any extent responsible for the differences in localization of the injected lymphocytes between prednisolone-treated and control recipients, an audit of radioactivity in all the tissues detailed in Fig. 1 and Table 1 was drawn up. For some organs such as the spleen, thymus, and mesenteric LN the total radioactivity was measured directly because the whole organ was inserted into a vial for gamma counting. For other organs such as the liver, lung, gut, and bone, where samples of the tissue were removed, total radioactivity was estimated by multiplying the mean percentage injected radioactivity per gram of sample by the mean organ weights of a “standard” 200-g rat (19). The results showed clearly that there was no significant difference between the two groups; at all time intervals the total recovery from both prednisolone and untreated groups was between 70 and 80% of the injected dose. At 24 hr after the injection of labelled TDL, the low LN recovery was fully compensated by the higher bone marrow and spleen recoveries. In particular the liver localization was lower in the treated than untreated rats. The conclusion that cell death was not responsible to any extent for the differences in distribution was fortified by two experiments. First, labelled cells from the bone marrow of treated and untreated primary recipients were retransferred into secondary recipients. Their distribution patterns were almost identical at 0.5,2.5, and 24 hr after transfer (not documented). These results must be treated with some caution, since only small numbers of
418
COX AND FORD 5 4
Bone (tibia)
‘\
3
‘\.,.-•
\
.\
l1.
2 1
\ o’“\o/o \
.
o-o\ 0.15
o-O-0 Blood
9
0.10
\
O/O\ O-------O
0.05
MesentericLymph
60 -
Node
Cervical Lymph Node 5040’ 0
50 ’
Aop>(’
40 ’ 0
.,./’
30’
30’ 20’
20’ I .
10’
lo-
I 6 5
10
15
20
1 25
b
1
5
10
15
20
25
Time after stopping Prednisolone (hours) FIG. 3. Localization of S’Cr-labelled lymphocytes in prednisolone-treated (0) (n = 6-7) and control (0) (n = 4-6) recipients after stopping the administration of prednisolone as described in the text. The values plotted are the mean 8 of the injected dose per gm of tissue. Standard deviations are recorded in Ref. (29). The approximate values for whole organs can be calculated by reference to Table 2 or Ref. (19).
lymphocytes could be recovered from the bone marrow of untreated rats for injection into secondary recipients. Second, in some recipients of labelled TDL, the prednisolone infusion was stopped after 24 hr and rats were killed 3, 6, 12, and 24 hr later. The high bone marrow localization started to decrease within 3 hr, thus increasing the blood level and later the LN localization (Fig. 3). Twenty-four hours after stopping prednisolone the localization in LN was greater than in the untreated rats, but even by this time the bone marrow localization in the prednisolone-treated rats, though still falling, had not reached the level of the untreated rats (Fig. 3). (9) Differential Effect of Prednisolone on the Traffic of T and B Lymphocytes Studies in humans (5) and guinea pigs (8) have suggested that the effect of corticosteroids in arresting lymphocyte traffic is greater on T cells than on B cells.
PREDNISOLONE
ARRESTS
LYMPHOCYTE
419
RECIRCULATION
This proposition was investigated in rats by exploiting Howard’s finding that T lymphocytes take up lo- 15 times more [ 3H]uridine in vitro than do B lymphocytes (22). TDL labelled in vitro with [3H]uridine were injected iv into prednisolonetreated or control rats which were killed 1 hr later. Autoradiographs of spleen, cervical LN, and bone marrow were exposed for several periods so that a time could be chosen at which there was a clear distinction between heavily labelled cells with areas of confluent grains and lightly labelled cells without confluence. In the control recipients 36-40s of the TDL that had entered the three tissues were lightly labelled, corresponding to the expected proportion of B cells (22) (Table 6). In the prednisolone-treated rats there was little difference in any of the three tissues, indicating that the traffic of B and T lymphocytes had each been perturbed in the same direction. There was a slightly increased proportion of heavily labelled T cells in bone marrow and correspondingly a slightly decreased proportion in the LN and spleen (Table 6), suggesting that prednisolone may exert a marginally greater effect on T lymphocyte entry into the bone marrow. DISCUSSION Previous studies of the impact of corticosteroids on the immune system have concentrated on describing changes in the weight and histology of lymphoid organs (23) and changes in lymphocyte concentrations in the blood (e.g., (5, 8)) and thoracic duct lymph (9, 24) after single or multiple doses of steroid. The novelty of the present study lies in following a population of recirculating lymphocytes in syngeneic recipients receiving a continuous infusion of a large (but apparently not harmful) dose of prednisolone. It was confirmed that corticosteroids kill lymphocytes in at least two compartments-the thymus and germinal centres in peripheral lymphoid tissue. However, neither of these compartments are in rapid equilibrium with the recirculating lymphocyte pool and therefore their complete destruction would not be expected to have an immediate effect on the number of recirculating lymphocytes. The present results firmly indicate that recirculating lymphocytes are not destroyed in a prednisolone-treated recipient. First, equal numbers can be accounted for in prednisolone-treated and control recipients. Second, the distribution of reTABLE
6
The Effect of Prednisolone Infusion on T and B Lymphocytes Prednisolone”
Cervical lymph node Bone marrow Spleen
Control
% Heavily labelledb
% Lightly labelled
% Heavily labelledb
% Lightly labelled
58.0 65.2 57.3
42.0 34.8 42.1
61.0 63.5 60.2
39.0 36.5 39.8
Note. [‘H]Uridine-labelled TDL injected iv 1 hr previously; 4000-5000 compartment. a Rat given an iv infusion of prednisolone 12-15 hr before injection. * Corresponds approximately to proportion of T lymphocytes (22). ’ Corresponds approximately to proportion of B lymphocytes (22).
cells were counted in each
420
COX AND
FORD
circulating lymphocytes starts to return towards that found in the control recipients within a few hours of stopping the prednisolone. And third, the excess of cells in the bone marrow of prednisolone-treated rats survive and migrate normally after retransfer to secondary recipients. It can be concluded that the profound deficit of small lymphocytes in the blood of prednisolone-treated rats, which affects both transferred and indigenous lymphocytes, is a consequenceof interference with the physiological migration pattern of lymphocytes. The retention of recirculating lymphocytes in the normal traffic areas has been suggested as the predominant factor in the lymphocytopenia (712). The present results support this notion in that they indicate prolonged retention of recirculating lymphocytes in the bone marrow, spleen, and lymph nodes of prednisolone-treated rats. Thus the high bone marrow localization 30 min and 2.5 hr after injection did not decreaseas long as the prednisolone infusion was maintained, in contrast to the control situation, in which a large decrease in marrow localization occurred between 2.5 and 9 hr after injection indicating a rapid transit time through bone marrow as argued by Rannie and Bell (25). Similarly, in the spleen the retentive effect of prednisolone is indicated by the maintenance of a high localization between 2.5 and 24 hr in contrast to the substantial redistribution from the spleen seen in the controls. In lymph nodes the transit time of lymphocytes recirculating from blood to lymph averages about 15- 18 hr for T cells as gauged by the modal recovery time from a thoracic duct fistula. Prednisolone treatment drastically inhibits this recovery although the site within LN at which cells are delayed is not known. In conclusion, prednisolone freezes the recirculation of lymphocytes within the tissues at which they arrive from the blood. The next question to consider is whether the sole effect of prednisolone on lymphocyte recirculation is retention in the tissues. The substantial reduction of the early (0.5 hr) localization of lymphocytes in the mesenteric LN suggests the possibility that prednisolone interferes to some extent with the influx of lymphocytes into LN, presumably by interfering with the adhesion of lymphocytes to and their migration across HEVs. The localization of lymphocytes in LN is directly proportional to the mean concentration in the perfusing blood (26); thus there is a greater deficit at 0.5 hr in the mesenteric LNs (39% of untreated) than would be expected from the blood levels (72% of untreated) and the integrated deficit in the blood over the relevant period (O-O.5 hr) was no doubt between 72 and 100% because the blood content of prednisolone and control recipients each started as 100% of the injected dose at time zero and after 0.5 hr continued to diverge. Clinching evidence for an inhibitory effect on lymphocyte influx came from the experiments on the popliteal LNs. Prednisolone treatment reduced the influx into the quiescent left popliteal LN to 26% of the untreated control, but the antigenically stimulated right popliteal LN showed no deficit at all (101% of the control, Table 5). Apparently the lymphocyte influx into the unstimulated lymph node is substantially inhibited by prednisolone, but antigenic stimulation 3 days previously exempts a LN from this inhibition. The significance of this exemption may be that it allows a stimulated LN to continue to recruit antigen-specific lymphocytes from the recirculating pool in the face of high levels of endogenous corticosteroids. This striking finding should be taken further by investigating the time relationship between the injection of antigen and the exemption of the LN from the inhibitory effect of prednisolone on lymphocyte influx. Two groups have found that the immunosuppressive effect of corticosteroids is most marked when given l-3 days
PREDNISOLONE
ARRESTS LYMPHOCYTE
RECIRCULATION
421
before antigenic stimulation; there is little or no suppressive effect when they are administered l-3 days after antigen (27-28). We suggest that established immune responsesmay be invulnerable to corticosteroid suppression because of the exemption of stimulated LN from inhibition of lymphocyte influx. Prednisolone did not produce any obvious structural change in HEVs apart from an increase in small granules staining with periodic acid Schiff. The autoradiographic analysis of the transit through the wall of HEVs suggestedthat prednisolone prolonged the time taken for lymphocytes to travel from the lumen to beyond the basement membrane. In A0 rats this process normally takes about 5- 15 min (Fig. 2 and unpublished data of W. L. Ford and M. E. Smith), but prednisolone treatment increases this time to more than 60 min. The difference is not because lymphocytes take a longer time to arrive at the LN from the blood in the prednisolone group; on the contrary the bulk of lymphocytes arrived more promptly in the treated group. Moreover, there was no significant difference in LN blood flow in the two groups. Lymphocytes passed between the endothelial cells of the HEVs in the prednisolone-treated rats but accumulated on the vascular side of the basement membrane as if unable to penetrate it (29). The bone marrow was the predominant site of the excess of lymphocytes in the prednisolone-treated group, in conformity with previous studies (8, 10, 12). The extraordinary rapidity with which this excessdeveloped (10 min after iv injection) argued that prednisolone facilitated the extraction of lymphocytes from the blood by vascualr endothelium in bone marrow. Thus prednisolone exerted opposite effects on the entry of lymphocytes into bone marrow and their entry into lymph nodes. The effects of prednisolone described in this paper may be primarily on lymphocytes themselves or primarily on other structures such as vascular endothelium, macrophages, and reticular structures. The exposure of mouse lymphocytes in vitro to hydrocortisone at 0.2 mg/ml for 4 hr did not affect their distribution in vivo after iv injection, but, not surprisingly, 2 mg/ml for 4 hr did impair their migration (30). In a thorough study more physiological concentrations of hydrocortisone have been found to inhibit the chemotaxis of rat lymphocytes in vitro with a gradation of effect from lo-’ to 10m3M (3 1). Our findings that (a) a single large dose of prednisolone had little effect on lymphocyte distribution, and (b) lymphocytes transferred from a prednisolone-infused rat to an untreated secondary recipient migrated normally, are both most easily explained by an indirect or secondary effect on lymphocytes, but other explanations are certainly possible. The traffic of lymphocytes is vitally important to both the initiation and development of immune responses(14, 32). The arrest of lymphocyte traffic as described here may contribute substantially to the immunosuppressive effects of corticosteroids, but its importance relative to the other possible mechanisms is difficult to judge at present. ACKNOWLEDGMENTS We wish to thank Mrs. N. Rattray and Mrs. T. Aslan for technical assistance.The work was largely supported by MRC Programme Grant G 977/455/B. J.H.C. held an MRC research studentship.
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422
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FORD
3. Dougherty, T. F., Physiol. Rev. 32, 319, 1952. 4. Dougherty, T. F., Berliner, M. L., and Schneebeli, G. L., Ann. N. Y. Acad. Sci. 113, 825, 1964. 5. Fauci, A. S., and Dale, D. S., J. Clin. Invest. 53, 240, 1974. 6. Van Dijk, H., Bloksma, N., Rademaker, P. M., Schouten, W. J., and Willers, J. W., Int. J. Immunopharmacol.
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