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Permeability of Rheumatoid and Normal Human Synovium to Specific Plasma Proteins.

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1550
PERMEABILITY OF RHEUMATOID AND
NORMAL ‘HUMAN SYNOVIUM TO
SPECIFIC PLASMA PROTEINS
J. RODNEY LEVICK
A method is described for the determination of
the permeability of the blood-joint barrier to specific
plasma proteins, using the ratio of protein concentration
in synovial fluid to that in plasma. The inadequacy of
the ratio per se as a direct index of permeability is
discussed. Permeabilities are evaluated for the normal
and rheumatoid human knee. Permeability increases in
the rheumatoid knee by approximately 6 times for
albumin and over 40 times for macroglobulins. The
effect of protein molecular dimensions upon permeability is analyzed. Permeability shows less dependence upon
solute dimensions in the rheumatoid knee than in the
normal knee, i.e., molecular selectivity is reduced.
From these data and synovial morphology, a twomembrane model of the blood-joint barrier is developed. The relative contribution of the component intima1 and endothelial layers to the total barrier is found to
depend upon solute dimensions.
The permeability of the blood-joint barrier to
plasma proteins determines the oncotic pressure of
synovial fluid. The difference in oncotic pressure
across the barrier is one of several factors regulating
flow into the joint space (1,2). Thus, changes in
permeability to plasma proteins may contribute to the
pathogenesis of rheumatoid and other inflammatory
synovial effusions (2). Yet synovial permeability to
plasma protein has been measured directly by only one
Supported in part by the Arthritis and Rheumatism Council
of Great Britain.
Address reprint requests to J. Rodney Levick, Senior
Lecturer in Physiology, St. George’s Hospital Medical School,
London SW17 ORE, England.
Submitted for publication November 20. 1980; accepted in
revised form June 11. 1981.
Arthritis and Rheumatism, Vol. 24, No. 12 (December 1981)
group (3,4); synovial permeabilities to specific proteins of known physical dimensions have never been
reported.
Many investigators (5-7) have measured the
concentration of specific plasma protein in synovial
fluid (C,) and plasma (Cp), and have intuitively adopted the fluid/plasma concentration ratio C F / C ~as a
direct measure of permeability. This paper demonstrates that CF/Cp is not a linear index of permeability. Also it depends on other variables in addition to
permeability; consequently, the use of CF/Cp as a
direct index of permeability is not justifiable. C&p
may serve as a nonlinear index of permeability, but
only if volume flow into the joint (equal to lymph
outflow) is constant, and only after correction of CF
for steric exclusion by hyaluronate. A method is
described, however, by which permeability can be
calculated from CF/CPand volume flow. The method
is applied to data for rheumatoid and normal human
knees; the relationship between permeability and solute dimensions is analyzed.
METHODS
Theory: Relationship between permeability and synovial fluidplasma concentration ratio. The permeability (P) of
a membrane of area (A) is, by definition, the diffusional flux
of solute (Js”) per unit concentration difference across the
membrane. The membrane that separates synovial fluid from
plasma consists of two layers in series, namely capillary
endothelium and synovial intima (Figure 1). For this composite “blood-joint barrier,” permeability is given by:
equation 1:
P = JsD / A (CpCp‘)
where C p is mean solute concentration in capillary plasma
and CF’ is “effective” solute concentration in synovial fluid.
PERMEABILITY OF HUMAN SYNOVIUM
1551
-
BLOOD-JOINT BARRIER
1-
[SYNOVIAL
. . . . . . . . . . . .
. .. .. . .
. . . . . . .
INTIMA~
I - [
PROTEI
(Jd
BLOOD
C A P ILLAR Y
. . .
FLUID
(Jv)
LYMPH
VESSEL
Figure 1. Diagram of protein and fluid exchange between plasma, joint cavity, and lymph vessels in the
steady state (highly schematic; thickness of endothelium greatly exaggerated). Protein passes from plasma
(concentration C), at rate Js across two barriers in series. namely capillary endothelium and synovial intima;
movement is mainly by diffusion. Fluid flows down pressure gradients from plasma into the joint cavity at
rate J,. Protein concentration in synovial fluid (C,) is determined by the relative rates of protein influx and
water influx. Because the system is in a steady state, fluid must also drain from the joint cavity at rate J,,
probably into the lymphatic system. Local synthesis of immunoglobulins, which becomes significant in the
rheumatoid joint (see text), is omitted for clarity.
The effective solute concentration exceeds the measured
concentration CEbecause solute is excluded from a fraction
of the solvent volume by the polysaccharide fibers of synovial hyaluronate (8,9). If the fractional available volume is
KAV,then
equation 2:
If a solvent flow occurs across the membrane, solute
not only diffuses across the membrane but is also “dragged”
along by the solvent (solute flux JsV). Kedem and Katchalsky (10) have shown by the methods of irreversible
thermodynamics that net solute flux Js is then given by:
equation 4:
CF’ = C p
/ KAv
The fractional available volume is given by the Ogston
equation (8):
equation 3:
KAV= exp (-nL (rf + re,)2)
where rf is hyaluronate fiber radius and L is fiber concentration in units of lengthlvolume. res is the Einstein-Stokes
diffusion radius of the solute.
where Jv is volume flow across the barrier, u is the solvent
drag or osmotic reflection coefficient ( I I ) , and C is mean
solute concentration within the barrier [approximately (Cp t
CF’)/ 2 (12)]. In the steady state, solute influx (Js) must also
equal the concentration of solute CF divided by the volume
influx, since, by definition, concentration is the ratio of
solute to volume (see Figure 1):
LEVICK
1552
equation 5 :
Cr = Js/Jv
Volume influx JV is net filtration from plasma and is determined by such factors as capillary blood pressure and
oncotic pressure (I). Substitution of equations 2 and 4 into
equation 5 leads to an expression that describes the relationship between permeability and the observed ratio CF/Cp
(represented by R):
equation 6:
biophysical details of the outflow system, since fluid is
sampled before outflow.) Thus, lymph flow JV can influence
synovial protein concentration CF, as is described by equation 7. Expressed nonmathematically , the lymph vessels
affect synovial protein level by influencing the rate of
washout of the joint cavity. Although lymph flow affects CF,
it does not affect the permeability of the blood-joint barrier.
Equation 7 is also applied below to data from rheumatoid joints. Because the assumption of negligible transport by bulk flow is, a priori, more dubious in such joints, the
values of Jv/(R-l - KAV-') should be regarded only as
approximations of permeability-surface area product PA; Jv/
(R- - KAV-I ) represents, strictly, total solute flux (by
diffusion plus bulk-flow) per unit transsynovial concentration difference. It will be shown that these values provide
important information regarding the physical processes by
which protein is carried into rheumatoid joints.
Data. C,JCp.The concentrations of specific plasma
proteins or protein fractions in plasma and in the fluid from
normal and rheumatoid human knees have been intensively
investigated in many previous biochemical studies (5-7.1420). Data from 13 sources are summarized in Table 1.
KAV. Fractional available volume in synovial fluid
may be calculated from Ogston's equation (equation 3).
Hyaluronate fiber radius rf is 3.5 x lo-' cm and L (cm/cm3)
is given by:
'
Equation 6 is not readily solved with the data available in the literature because synovial u for specific proteins
has never been reported. However, a simpler equation,
which can be solved, is obtained if solut? transport by
solvent drag [the bulk flow term Jv(l - v)C] is neglected.
Data exist that provide some justification for this simplification. Simkin and Pizzorno (3,4) found PA for plasma protein
to be 0.008-0.011 ml/min for the normal human knee. The
difference in total protein concentration across the normal
bamer averages 49 mg/ml (Table 1); hence, the diffusional
protein flux JsD is 39 x lo-* -54 x lo-' mglminute. The
volume flow Jv into the normal knee is about lo-' ml/minute
(2). If we assume u is 0.8 or more ( I ) , the protein flux due to
solvent drag (Jsv) is 1 9 x lo-.' mglminute. Thus, diffusion
accounts for at least 77-83% of plasma protein flux across
the normal blood-joint barrier.
If the relatively small nondiffusional component of
protein influx is neglected, a simple relationship between
steady-state concentration ratio R (= CF/Cp)and permeability is derived. Substitution of equations 1 and 2 into equation
5 gives:
equation 7:
[An analogous expression was first derived by Renkin (13)
for lymph/plasma concentration ratios, where KAv is unity.]
It can be seen that the steady-state fluid/plasma concentration ratio R is a hyperbolic rather than linear function of
permeability. R depends on volume flow and fractional
available volume as well as on permeability. Thus, the ratio
R cannot serve as a direct index of permeability. Equation 7
does, however, enable permeability to be calculated from R
for the normal joint.
Although equation 7 is derived solely from consideration of influx into the joint cavity from plasma, it describes
the steady-state situation, in which influx equals efflux.
Therefore, Jv also represents fluid outflow, while protein
efflux must equal JvCr;. Fluid and protein probably drain
from the joint cavity via intercellular channels into lymph
vessels, which lie deep to the intima and do not open directly
into the joint cavity; thus, JV also equals lymph flow (Figure
I). (It is stressed, however, that the above analysis is
independent of any assumptions as to the anatomic or
L =
[HA] x NA X 10.3
lo00 x 379
X
lo-'
where [HA] is hyaluronate concentration in gdliter; 379
daltons is the molecular weight of the component disaccharide unit of length 10.3 x lo-' cm (9); and NA is Avogadro's
number (6.02 x loz3).Hyaluronate concentrations in human
synovial fluid were obtained from the literature (Table 1);
they average 3 g d i t e r in the normal knee and 1 gdliter in
the rheumatoid knee.
J v . Net volume flow from plasma into the normal
knee (Jv, equal to lymph outflow) is -lo-' ml/minute (2).
Flow through the rheumatoid knee is estimated to be
-2.10-2 ml/minute. This value was obtained by calculation
of the flow which causes the calculated permeability to the
smaller plasma proteins to lie in the range measured directly
by Simkin and Pizzorno (4) for total synovial protein. The
increase in fluid flow into (and therefore lymph flow out of)
the rheumatoid joint tends to minimize the rise in protein
concentration CF and renders CF in isolation a misleading
guide (an underestimate) to the change of permeability in
rheumatoid synovium.
RESULTS
Table 1 summarizes the fluid/plasma concentration ratios for various plasma proteins. Not all sources
reported the CF and Cp values from which the published ratios were calculated; consequently, some of
the values of R in the table differ slightly from the ratio
of the quoted CF and Cp values. The fluid/plasma
concentration ratio is always less than unity and
PERMEABILITY OF HUMAN SYNOVIUM
1553
Table 1. Concentrations of plasma proteins and hyaluronate in synovial fluid from normal and rheumatoid human knees*
....
-_
-.
-__
___..
Molecular
Normal fluid, mg/mlS.
Rheumatoid fluid, mg/ml
-__.
.
weight
Solute (ref)
(KdV
Fluid
Plasma
CF/Cp
Fluid
Plasma
c
JC p
~.
_____
- __
__
-_
Total protein (6.14-16,19,20)
I9.?)<. 2)
67.7(I .7)
0.28
50.0(4.8)
70.0(35)
0.71
a,-globulins (5.14-16.19)
-44
1.3(0.1)
4.1(0.4)
0.32
?.5(0.6)
4.0(0.3)
0.63
44. I
0.7
Orosomucoid (5,6,17,20)
0.8
1.3
0.65
0.31
Albumin (5.14-16,19,20)
69
12.0(1.4)
32.7(3.0)
0.37
i9.1(1.3)
29.2(0.2)
0.65
Transfenin (6,7)
74-90
0.24
1.4
2.3
0.62
Prothrombin-proconvertin (5)
68.5-74
0.10
0.36
P,-globulins (IS)
2.7
10.5
0.26
Haptoglobin (5,17,18,20)
100
0.09
I .8
0.05
0.9
2.4
0.38
IgG (6.7.20)
-150
0.13
10.3
20.1
0.67
y-globulins (5.14-16.19)
160
2.4(0.4)
I1.3( I . 5 )
0.21
Il.O(O.6)
17.0(2.1)
0.65
Ceruloplasmin (6)
160
0.16
0.53
IgA (7,20)
160-400
1.6
3.6
0.47
a2-globulins (5,7,14-16.19)
loo-820
I . l(O.2)
7.9(0.3)
0.14
3.2(1.2)
6.9(2.6)
0.46
a2-macroglobulin (6)
820
0.033
0.35
P2-globulins
340-1 ,OOO
0.5
4.9
0.09
(? inc. fibrinogen) (15)
IgM (6.7)
900-1,OOO
0.045
0.5 1
1.44
0.43
Hyaluronate (21-23)
-6,000
3.0
1
__
__
__
-_
-_
* Data are averages of concentrations presented in the references in parentheses.
t Molecular weights are from these references and from F. W. Putnam’s The Plmrnci Proteins (33).
$ Numbers in parentheses are standard errors of the mean; the presentation of the data do not enable SE to be calculated in all cases. Normal
fluid was largely sampled postmortem.
~
~
~
-
~
~
~~
declines as protein dimensions increase. These low
ratios might be due to steric exclusion of plasma
protein by synovial hyaluronate (24), i.e., represent
the fractional available volume in synovial fluid. This
proposition is tested in Figure 2, which shows fractional available volume plotted against Einstein-Stokes
diffusion radius of solute (re,), for fluids containing
various concentrations of hyaluronate. On the same
graph, the ratios CF/CP (normal knee) are plotted
against res. The observed ratios are considerably
smaller than can be accounted for by steric exclusion
by hyaluronate at physiologic concentrations (2-4 gm/
liter); a hyaluronate concentration of at least 12 gm/
liter would be required to produce most of the observed ratios. Similarly, the ratios for rheumatoid
knees cannot be explained by the rheumatoid concentration of hyaluronate (-1 gmfliter). The low ratios
therefore do not represent an equilibrium state, but are
caused by the low permeability of the blood-joint
barrier to plasma protein.
Permeability-area products (PA) for specific
plasma proteins were calculated from the ratios in
Table 1 via equation 7 and are tabulated in Table 2. In
the normal knee, PA for plasma albumin (MW 69 Kdj
is 7 X lop3cm3/minute. PA declines as protein molecular size increases, falling to 0.4 x lop3cm3/minute for
u2-macroglobulin (MW 820 Kd).
In the rheumatoid knee, the transsynovial flux
of each protein per unit concentration difference (ap-
~
~
proximate PA, see Theory) is increased. The relative
increase is greater for large proteins than for small
proteins. For example, PA for albumin increases by a
factor of 6.36 in the rheumatoid knee, while PA for a?-
es ( n m )
P
Figure 2. Relationships between fractional available volume (KAV)
and Einstein-Stokes diffusion radius of solute (rCJ in solutions
containing various concentrations of hyaluronate. The number
against each curve represents hyaluronate concentration in g d i t e r .
Curves were calculated from equation 3. The solid triangles show
the observed fluidplasma concentration ratios (CF/Cp) for the
normal human knee, taken from Table 1 . The experimental data are
not distributed around the curve for hyaluronate at physiologic
concentration (-3 g d i t e r ) .
LEVICK
1554
.
.-C
E
\
E
Q
Q
I
.I
1
I
10
1
100
Figure 3. Relationships between permeability-surface area product
(PA) and Einstein-Stokes diffusion radius (re,) for the blood-joint
barrier of the human knee. Data are from Table 2 (solid symbols)
and references 3 and 4 (open symbols); circles represent data from
the normal joint and triangles data from the rheumatoid joint. The
plotted values are total solute flux per unit transsynovial concentration difference, and, in the case of rheumatoid joints, should be
regarded only as approximations of true PA (see Theory). Solid lines
are lines of best fit for the protein permeabilities of Table 2 (linear
regression analysis): the steeper of the two lines drawn through the
rheumatoid data is the line of best fit if data for y-globulin, IgG, and
IgM are excluded (because of local synthesis of these proteins by
rheumatoid synovium). The parallel, dashed lines have a slope of - 1
and represent the decline in PA that would result purely from the
decline of free diffusion coefficient as solute radius increases.
macroglobulin increases by 41.3. The quantitative
error inherent in the adoption of Cp/Cp as a direct
measure of permeability is well illustrated here;
whereas PA for albumin increases 6.36 times in the
rheumatoid knee, the albumin concentration ratio CF/
Cp increases only I .76 times. The large increases in PA
for plasma proteins in rheumatoid arthritis contrast
with the slight reductions in PA for small solutes, such
as urea, reported by Simkin and Pizzorno (4).
The high concentrations of nonimmune proteins
in rheumatoid fluid are caused by the raised permeabil-
ity of the rheumatoid blood-joint barrier. An alternative potential cause of high protein levels, namely
lymphatic obstruction (reduction of term Jv in equation 7), is discredited by the data, because l ) large
proteins show a greater rise in concentration than do
small proteins (molecular selectivity reduced), whereas lymphatic obstruction would elevate the concentrations of all proteins by an equal factor (equation 7);
and 2) lymph flow appears to increase rather than
decrease in the rheumatoid joint (see Methods).
The relationship between permeability and solute radius for both normal and rheumatoid joints is
illustrated in Figure 3. Also shown are values of PA for
small solutes (THO, urea, urate, creatinine, glucose,
sucrose) as reported by Simkin and Pizzorno (3,4).
Data are plotted on logarithmic axes to facilitate
comparison with the change in free-diffusion coefficient. The free-diffusion coefficient (D) of a solute is
inversely proportional to the Einstein-Stokes radius ID
cll/res or log D c1 - 1 x log res (27)l. Thus, on a log PA
versus log res plot, a line of slope - 1 would indicate a
decline in permeability which is wholly explicable by
the decline in free-diffusion coefficient with solute
size. For the normal blood-joint bamer, however, a
line relating log PA and log re, for solutes of radius
0.5-3 nm is constrained to have a slope steeper than
-1, i.e., permeability declines more steeply than can
be explained by the change of free diffusion coefficient. This phenomenon has been termed “restricted”
diffusion (28) and indicates that solutes of radius 0.5-3
nm do not diffuse freely within the normal blood-joint
barrier. The decline in permeability as re, increases
above 3 nm (protein data) is less dramatic but nevertheless appears to be slightly greater than that attributable to a decline in free-diffusion coefficient. The
regression line relating log PA and log res for the
protein data has a slope of -1.23 (or -1.61 if the
prothrombin and haptoglobin data are omitted, see
below). These slopes are not statistically significantly
different from - 1, however (Student’s r-test).
In regard to the rheumatoid knee, there is no
evidence for restricted diffusion; the slope relating log
PA to log res is no steeper than -1 throughout the
range of molecular sizes. Indeed, the regression line
for the protein data has a slope of only 0.09, which is
significantly less steep than -1 or than the slope for
the normal blood-joint barrier. Thus, although the
permeability to each protein is increased in rheumatoid arthritis, the molecular selectivity of the bloodjoint barrier (i.e., decline in permeability per unit
increase in solute radius) is considerably reduced.
PERMEABILITY OF HUMAN SYNOVIUM
1555
Table 2. Permeability area products (PA) for the blood-joint barrier of normal and rheumatoid knees,
calculated from CF/Cp
Normal knee
Solute
a,globulins
Prothrothrombinproconvertin
Albumin
Transferrin
Orosomucoid
Haptoglobin
Ceruloplasmin
y globulins
IgG
IgA
macroglobulin
pZ globulins
IgM
cr2
PA, pl/min
P(X 10
cm/min)t
Rheumatoid kneet
PA. pl/min
3.0
5.17
I .87
38.2
-3.4
3.6
3.6-4.3
3.9
4.6
4.5-5.6
5.6
5.6
5.7
9. I
10.8
11.0
1.14
6.96
3.53
5.24
0.54
2.13
3.33
I .68
0.38
2.25
0.66
0.41
2.51
I .27
1.89
0.19
0.77
I .20
0.61
0.14
0.81
0.24
11.8
44.3
39.9
46.8
13.4
27.6
Solute radius,
rcs. nm*
59.9$
69.5$
22.0
15.7
49.7$
* r,,, the Einstein-Stokes diffusion radii are from references 9, 25, 26.
t Synovial area (A) in the normal adult human knee = 277 cmz (40). P for rheumatoid joints has not
been calculated, because no value for A appears to be available. The PA values calculated for the
rheumatoid joints prove to be only approximations of the true diffusional permeability of the
rheumatoid bamer, because transport by bulk flow is not negligible (see Discussion); the listed values
are strictly total transsynovial solute flux per unit concentration difference between plasma and
synovial fluid.
t. Probably an overestimate due to local synthesis of immunoglobulin by infiltrated cells in the
rheumatoid joint (see text).
A number of anomalous PA values are evident
in Table 2. PA values for haptoglobin and prothrombin-proconvertin are unusually low compared with
those for other proteins of similar molecular radius.
The electrophoretic mobility of haptoglobin is relatively high for its molecular weight, indicating a relatively
high negative charge at physiologic pH (25). The low
permeability of the barrier to this densely charged
macromolecule might therefore indicate the existence
of a high negative-charge density in a component of
the barrier. There is considerable evidence that molecular charge as well as size affects the permeability in
other vascular barriers (29-32). Prothrombin, though
of molecular weight 68.5-74, Kd (331, behaves as a
larger molecule of equivalent molecular weight 90-100
Kd on Sephadex column chromatography (34) and
appears to behave as an even larger solute in the
blood-joint bamer.
In the case of rheumatoid joints, the apparent
PA values for y-globulin and IgG are unduly high
compared with other proteins. This may be attributed
to local immunoglobulin synthesis by infiltrated cells
(6). Perhaps, the apparently high permeability to IgM
might be similarly explained.
DISCUSSION
The blood-joint barrier has been regarded as an
essentially passive obstacle composed of two rnembranes in series: the synovial intimal layer and the
underlying capillary endothelium (4). The question
arises whether the known properties of the components of these layers (Figure 4) are consistent with the
relationships between permeability and solute dimensions described in the Results section. To answer this
question, we must first evaluate the relative contributions of the two layers to the normal total resistance to
solute exchange (resistance = l/PA). Such an evaluation is possible from morphometric data. The procedure may be justified by the work of Casley-Smith,
O’Donoghue, and Crocker (35), who showed that
morphometnc calculations yield parameters close to
those determined by direct physiologic experiment for
the blood-jejunal barrier.
From Fick’s law of diffusion, the ratio of resistances of the layers to a very small solute, whose
diffusion is negligibly restricted within either layer, is
(Al/lI)/(AJL), where A is equivalent pore area and 1 is
pathlength in membranes 1 and 2. For an extracellular
1556
LEVICK
ICG
ICC
1
f
Synovial fluid
1
Intima
j- Endothelium
Plasma
v
ICJ
F
Figure 4. Schematic diagram of the blood-joint barrier, based on electron micrographs (not to scale). ICG =
intercellular gel. ICC = interstitial channel of Casley-Smith and Vincent (48). V = vesicle in lumenal aspect of
capillary endotheliurn. ICJ = intercellularjunction, between two endothelial cells. F = fenestral membrane.
The intimal layer is represented for simplicity by a single layer of cells and interstitiurn, but is in reality often
2-3 cells deep over the subintimal capillary plexus. Endothelial basement membrane is not separately
represented on this diagram, being treated as part of the extracellular matrix for the purposes of the present
analysis.
solute, AJA2 may be estimated from the ratio of the
area of intimal intercellular matrix to area of endothelial “pores,” provided that the fractional content of
impermeable matter is not grossly dissimilar in the two
permeable regions.
The area of endothelial pores is taken to be that
of intercellular junctions [-0. 1% of endothelial area
(36)] plus fenestrae, which are present in roughly half
the capillaries and occupy 0.06% of the area of such
vessels (2,37). Endothelial area in the subintimal capillary plexus at 10 pm depth may be calculated from the
mean intercapillary distance [--120 prn (38,39)] and is
-15.7% of synovial area, or 43.5 cm’ for the human
knee [synovial area 277 cm’ (40)]. This calculated
value is less than 1/100th of the estimate by Simkin and
Pizzorno (3), which was based on capillary morphology in skeletal muscle; this brings into doubt their
conclusion that aggregate endothelial resistance to
small-solute exchange is negligible. (The issue highlights the urgent need for better, quantitative data
regarding the synovial area and microcirculation.)
The area of intimal matrix is 7% of synovial
area [rabbit knee (2)l. If a similar percentage applies to
human synovium, as published electron micrographs
seem to show, then matrix area is 19.4 crn2 in the
human knee, cf., the value of 0.57 cm2 inferred by
Simkin and Pizzorno (3).
The depth of the intimal barrier over the superficial capillary plexus averages -10 p.m. The thickness
of the endothelial barrier is far less (-0.1 pm for the
intercellular junction and 3-5 nm for the fenestral
membrane). Substitution of these morphologic data
leads to the conclusion that
into the ratio A1/i2/A2/i1
the intimal layer accounts for 52-78% of the total
resistance of the normal blood-joint barrier to the
diffusion of a small extracellular solute. The capillary
wall accounts for only 114-1/2 of the total resistance, in
spite of its small pore area, because the pathlength
across the fenestral membrane is only 1/3,000 of that
across the intima.
The theoretical effects of solute dimensions on
the permeability of such a two-layer system may now
be calculated and compared with the data (Figure 5 ) .
First, the simplest system will be considered: many
small, uniform endothelial pores and a uniform extracellular glycosaminoglycan matrix. Endothelial pore
radius is estimated to be 5 5 nm, because such channels generate an osmotic reflection coefficient of 20.8
PERMEABILITY OF HUMAN SYNOVIUM
h
.-C
E
\
E
u
Q
a
Figure 5. Comparison of theoretical and experimentally determined permeabilities of the blood-joint barrier (PA) to solutes of
increasing molecular rddius (rer). Data are represented by circles
(normal knee) and triangles (rheumatoid knee) as described in the
legend to Figure 2. Curves A to D are theoretical relationships
between the permeability-surface area product and molecular radius
for a barrier composed of two membranes (intirnal interstitiurn and
capillary endothelium) in series. Curve A (slope -1) describes a
b a m e r whose “pores” are sufficiently large not to restrict the
diffusion of solute. Curve B describes a bamer containing restrictive
5 nm radius transendothelial channels (or a dense fiber matrix) in
series with interstitial glycosaminoglycan gel equivalent to 5 gm
hyaluronatefliter. Curve C is as for B plus a system of 30 nm radius
vesicles transporting water and solutes across the capillary wall at
2.5 pYminute. Curve D is as for C plus a system of 71 nrn radius
channels permeating the intimal gel phase.
for plasma albumin (1,41). Solute diffusing through
such a pore is “restricted” by steric exclusion effects
at the pore entrance and by friction within the pore.
The degree of restriction is proportional to solute
radius, as described by Pappenheimer (28,36) whose
equations (see Appendix) were used to construct
curve B of Figure 5 . [A similar curve may be obtained
if endothelial permeability is represented by a dense
fiber matrix rather than cylindrical pores (42).] The
extracapillary interstitial barrier is considered to re-
1557
strict diffusion to a degree not less than that of 5 gm
hyaluronate/liter (43). The potential importance of
molecular sieving by such a layer has been stressed by
Nettelbladt and Sunblad (44). Restriction to diffusion
through such a matrix was calculated from the equations of Ogston (8) and Ogston, Preston, and Wells
( 4 9 , as described by Curry and Michel (46) (see
Appendix). Curve B of Figure 5 shows that the theoretical curve for such a composite barrier describes the
small solute data of Simkin and Pizzorno (3) reasonably well but deviates increasingly from the experimental data as molecular dimensions increase. This is
due mainly to the sharp decline in endothelial permeability as solute dimensions approach pore dimensions.
The low but finite permeability to solutes such
as the globulins, which are far larger than the “small”
pores, demonstrates the existence of an additional
pathway of wider dimensions across synovial endothelium. Ultrastructural studies of the transsynovial flux
of ferritin indicate that protein can cross the capillary
wall in cytoplasmic vesicles (47), of radius -30 nm.
When the theoretical curve is recalculated to incorporate a vesicular turnover of 2.5 x 10 - 3 cm3/min and to
allow for the vesicle excluded-volume effect as formulated by Garlick and Renkin (32), the new curve (curve
C, Figure 5 ) now fits the experimental data more
closely. Even so, the calculated restriction to the
exchange of very large proteins is still too great. This
is because the interstitial matrix begins to restrict the
diffusion of very large solutes to a considerable degree. Casley-Smith and Vincent (48), however, have
recently described aqueous channels of radius 71 nm
that permeate the intimal matrix. When diffusion and
solvent-drag through these large, relatively unrestrictive channels are included in the calculation of intimal
solute flux, a further improvement between theory and
data results (curve D of Figure 5). Therefore, recognized components of the normal blood-joint barrier
(Figure 4) can together provide a reasonably satisfactory explanation of the permeability-solute dimension
relationship presented in the Results section.
By use of the above analysis, the relative contributions of the endothelial and intimal layers to the
overall barrier can be calculated for solutes of various
sizes (Figure 6). For small lipid-insoluble solutes such
as electrolytes, urea, and glucose, the intimal layer
contributes more to the net barrier to exchange. For
larger solutes such as the principal oncotic agent,
plasma albumin, the barrier resides mainly in the
endothelial layer, because the small pores or dense
LEVICK
1558
1
Figure 6. Relationship between the molecular radius of solute and
the relative contributions of capillary endothelium and synovial
intima to the total resistance (IIPA) of the normal blood-joint barrier
to solute exchange. The relative contribution of each layer to the
resistance to diffusion of a small, negligibly restricted, extracellular
solute was calculated from morphometric data (see text). The
changes in contribution with increasing solute size were calculated
from the equations for restricted diffusion and steric exclusion (scc
Appendix), applied to endothelium perforated by 5 nm radius
channels and containing 30 nm radius vesicles and to intimal
interstitiurn permeated by 71 nm radius channels. Note that for
albumin, the chief oncotic constitutent of plasma, the major barrier
is capillary endothelium; but, for macroglobulins, the intima begins
to dominate the resistance to exchange.
fiber matrix of this layer restrict the exchange of such
solutes far more than does the intimal matrix. For very
large solutes such as IgM, however, the intimal layer
grows to dominate the net resistance to exchange,
because the growth in interstitial resistance to diffusion exceeds the decline in vesicular transport across
endothelium.
None of the above theoretical curves (B, C, or
D) adequately describe the permeability data for the
rheumatoid joint. This might be expected, because the
curves are derived for normal synovium. The rheumatoid data are, however, reasonably well fitted, at least
for solutes up to the dimensions of small proteins [data
of Simkin and Pizzorno (4)], by a line of slope - 1 (line
A). This implies that the diffusion of such solutes
across the combined endothelial-intimal barrier is not
significantly restricted in the rheumatoid synovium.
There are three possible explanations of the unrestrict-
ed diffusion of small solutes and increased permeabilit y to large solutes: 1) the existence of wider channels
than normal in the “tightest” component of the barrier, namely endothelium [for example, large interendothelial gaps such as those described by Schumacher
in chemically inflamed synovium (4911; 2) a reduction
in endothelial fiber matrix density; or 3) a greatly
increased rate of vesicular transport.
Although permeability to large solutes increases in rheumatoid synovium, there is a small
reduction in the permeability of the combined bloodjoint barrier to small solutes (e.g., urea), as demonstrated by Simkin and Pizzorno (4). As these authors
point out, this apparent anomaly is readily resolved by
the different physical effects of channel width compared with channel length or total number of channels
(i.e., membrane area). An increase in channel width
increases permeability to large solutes more than to
small solutes (see Appendix equations) and thereby
reduces the molecular selectivity of the membrane. An
increase in membrane thickness (channel length) or
reduction in membrane area (number of channels)
reduces permeability to large and small solutes to an
equal degree and does not affect the molecular selectivity of the membrane. It has, therefore, been suggested (4) that the reduced permeability to small
solutes may be due to an increase in pathlength (due to
hypertrophy of the intimal layer) and/or a decrease in
endothelial surface area (due to obliterative vasculitis).
If the number of large endothelial gaps or the
rate of vesicular transport increase in rheumatoid
synovium, then the contribution of bulk flow of fluid to
the net transport of protein may be expected to
increase (because u declines), especially for the largest
proteins. The data in Table 2 allow this supposition to
be tested. If protein transport across rheumatoid synovium were purely by diffusion, then the tabulated
values of Jv/(R-’ - KA” -’) (total flux per unit concentration difference) would equal PA. In this event, the
slope relating log PA to log res would have a minimum
slope of - 1 (i.e., - 1 or a more negative value). The
slope of the log-log plot for the protein data, however,
is 0.09 (or -0.67 if immunoglobulins are omitted from
the regression analysis). Therefore, transsynovial flow
contributes significantly to the transport of protein
into the rheumatoid joint. The above considerations
also confirm the suspicion that equation 7 at best
yields only a rough approximation of the true diffusional permeability to protein in rheumatoid joints (see
Theory ) .
PERMEABILITY OF HUMAN SYNOVIUM
APPENDIX
1559
(PA),,
The equations from which the theoretical
curves of Figures 2 and 5 were calculated are as
follows.
Net permeability (PA). For two membranes in
series:
where subscripts C and S refer to capillary and synovial intimal barriers, respectively. PA for a small, negligibly restricted solute (THO) is - I cm3/minute (3).
This leads to (PA)c = 2.08 cm3/minute and (PA)s =
1.92 cm3/minute for THO (see morphologic data in
Discussion).
Capillary permeability (PA)c. This is the sum of
permeability due to "small pores" (PA),, and that due
to vesicular transport (PA),. (PA),, for a solute X is a
fraction of that for water because of the change in free
diffusion coefficient (a l/res) and restriction of that
diffusion coefficient, i.e.,
=
THO
4'
2.08 x r
x rcrL
X
+THO
+
where is the function for restricted diffusion through
cylindrical pores of radius rp as developed by Pappenheimer (28,36):
+ = (l-re5/r,)2[1-2. I(r.=\/r,) + 2 . ~ ( r ~ ~ / r ~ ) 3 - o . 9 5 ( r ~ , / r ~ ) ~ ]
Permeability due to transport by vesicles of radius r,
at V ml/minute is given by Garlick and Renkin (32) as:
(PA),'
=
Synovial interstitial permeability (PA)s. This is
the sum of the permeability of the gel phase (PA), and
that due to aqueous interstitial channels (PA)i,. Representing interstitial gel by a hyaluronate fiber matrix:
2.
3.
5.
6.
7.
8.
10.
where restriction to diffusion within the gel phase is
given by the function of Ogston et al (45):
'
The "permeability" due to aqueous interstitial
channels that occupy -0.76% of matrix (48) is given
by:
x--
+'
+ Jv(1
- ~ rx)Cx
(cp- c,..)'
1. Knight AD, Levick JR: The control of fluid movement
12.
( d J M + rcd'l'
THO
x
REFERENCES
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f(0PW) = e
*
(The term permeability is, strictly, inappropriate here,
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wx, the solvent drag coefficient for solute X ,
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reciprocity. Lateral diffusion down concentration gradients between the gel phase and the aqueous channels
was not modeled and would further reduce the slope of
curve D in Figure 5.
9.
V(I-rc,'/rv)3
1.92 x 0.76 x 10
+'"O
4.
(PA),,'
==
13.
14.
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