splenic extravasation (long)

Aaron Warnock awarnock at cmgm.stanford.edu
Thu Feb 16 19:31:20 EST 1995

In article <9502161255.AA23618 at popserver.jr.cc.gettysburg.edu>,
Ralph A. Sorensen <rsorense at GETTYSBURG.EDU> asks:

>In preparing a lecture on the functional anatomy of lymphoid tissues,
>I have encountered a question that leaves a hole in my notes: how does
>extravasation of lymphocytes occur in the spleen? All of my sources
>wax eloquent on the role of the HEV in lymph nodes and unencapsulated
>lymphoid tissue, but the only reference I find to the spleen is that
>there is no HEV!  To further my confusion, while it is no problem for
>lymph node lymphocytes to return to the circulation via the efferent
>lymphatics, how do splenic lymphocytes return from the white pulp?
>Could someone help me out and/or point me to a reference? Thanks.
>Ralph A. Sorensen
>Biology Department, Gettysburg College
>Gettysburg, PA 17325           
>rsorense at gettysburg.edu             
>717-337-6168 (voice)  717-337-6157 (fax)       

"Holy cow!" I thought when I saw your post. Here I've been working in
the field for quite awhile without ever really thinking about this.
It's a good question, and there in fact are people who work on it (see
below), but not many.  Most of the known information is of the "while
we were doin' some other more important stuff in the lab, we looked"
type, as so it's mostly negative. You know,the "I checked out this or
that adhesion molecule and it doesn't seem to be all that important."
But basically, you're right, there's no HEV and not much known about
mechanism, however, the homing does seem specific and unique.

Anyway, by intravital microscopy, one can see that extravasation
happens through vessels (microvasculature-->venules in the marginal
zone/marginal sinus), but that's about the only comforting thing seen.
There is also the basic dumping of blood from arterioles into the MS.
To our knowledge, no anti-adhesion molecule antibody treatment, either
on pretreated labeled leukocytes or by IV injection against candidate
endothelial structures, has resulted in significant blockade of homing
or extravasation in spleen. Thus, neither the structures nor the
mechanisms of recognition for splenic trafficking are known. The only
published data that were aware of that regarding mechanism, is G.
Kraal's data with neuraminadase, suggesting a sialic acid requirement,
as well as speculation of a role of macrophages and other MZ residents
in leukocyte transmigration.

It is interesting to note that carbohydrate also plays a role in the
removal of RBC's in the splenic scavenger system. Additionally,
pertussis toxin has no demonstrable effect on the homing/extravasation
in spleen, suggesting an important difference from known mechanisms in
peripheral and mesenteric nodes, as well as Peyer's patches. In these
lymphoid tissues the lymphocytes require an "activation" signal that
results in the up-regulation of secondary adhesive structures,
primarily by integrin affinity modulation. This appears to be by
unknown g-protein linked receptors (which are pertussis toxin
sensitive). Does this mean a distinct signalling pathway in spleenic
trafficking? Or does it mean that extravasation there is simpler, and
more passive (especially 'cause the spleen gets more traffick than all
the other lymphoid organs combined!)? Does pertussis toxin affect the
transit from the MZ to the PALS, even if it doesn't effect initial
localization? Are there subsets that use distinct mechanisms, which
have been missed by gross homing studies? What is the role of
macrophage/lymphocyte interaction in the MZ?

Why haven't traditional methods led to identification of the molecular
players in spleen? Well, mostly 'cause the Stamper-Woodruff type of
frozen section assay for leukocyte binding is just pretty darn messy
with spleen tissue, and that makes screening for blocking antibodies
difficult. Other functional assays are not exactly obvious (for large
scale screening). So it's not that people wouldn't be interested, they
are; it's just that other things have been significantly easier to
deal with. I doubt if it'll be long though, until we know more. I've
heard a few things here and there, but since this isn't my pet (t)issue,
I'm waiting like you for someone else to figure it out.

As far as how cells exit, that is not clear. 10% of transmigrating
lymphocytes reach the white pulp, and spend an average of 4-5 hrs
there. What it takes to keep them there is probably dependent on
interaction with APC (speculation), and nothing is known about what
drives them to decide to leave. The path of exit has somewhat been
looked at. Basically, there are small "deep lymphatic vessels" which
pipe them out to lymph. Doesn't mean there aren't other ways of exit,
for example back the way they came...I point you towards one study in
particular (Pellas and Weiss).

But hey, EXCELLENT QUESTION. It's pretty important stuff, and once
again, we know little about it.

Hope some of this helps! Later,


Please find relevent stuff below (with sections of abstracts):

 Kraal G; Hoeben K; Breve J; van den Berg TK.
      The role of sialic acid in the localization of lymphocytes in the spleen.
    Immunobiology, 1994 Feb, 190(1-2):138-49.
      ABSTRACT available.  (UI 94364622)

 Kraal G.
     Cells in the marginal zone of the spleen.
   International Review of Cytology, 1992, 132:31-74.
     (UI:  92210257)
     Pub type:  Journal Article; Review; Review, Academic.

    ... It is this combination of relatively sessile cell
    populations and the continuous influx and passing of bloodborne
    immunocompetent cells that turn the marginal zone into a dynamic area,
    particularly apt for antigen processing and recognition. ......
    B memory cells
    then migrate into the PALS and present antigen to T cells. The marginal
    zone therefore functions not only as an area of initial filtration and
    phagocytosis of antigens from the blood, but also as a site of lymphocyte
    emigration. Some of the incoming T and B lymphocytes in the recirculating
    pool enter the white pulp from the marginal zone. The underlying force and
    selective molecular mechanisms that guide this migration are unknown. Both
    B and T lymphocytes recirculate through the outer PALS area on their way to
    the follicles and the inner PALS, respectively.
 Pabst R; Westermann J.
      The role of the spleen in lymphocyte migration.
    Scanning Microscopy, 1991 Dec, 5(4):1075-9; discussion 1079-80.
      ABSTRACT available.  (UI 92342924)
      Pub type:  Journal Article; Review; Review, Tutorial.

 Pabst R; Westermann J.
      The unique role of the spleen and its compartments in lymphocyte
    Research in Immunology, 1991 May, 142(4):339-42.
      (UI:  92021759)
      Pub type:  Journal Article; Review; Review, Tutorial.

Willfuhr KU; Westermann J; Pabst R.
       Absolute numbers of lymphocytes subsets migrating through the
       compartments of the normal and transplanted rat spleen.
     European Journal of Immunology, 1990 Apr, 20(4):903-11.
       ABSTRACT available.  (UI:  90269346)

Schmidt EE; MacDonald IC; Groom AC.
       Interactions of leukocytes with vessel walls and with other blood cells,
       studied by high-resolution intravital videomicroscopy of spleen.
     Microvascular Research, 1990 Jul, 40(1):99-117.
       (UI:  90377011)

    ...Mean numbers of marginated lymphocytes in
    venous vessels ranged from 0.1 to 4.5 per 1000 microns 2 of wall surface,
    and speeds of rolling from 11 to 20 microns/sec. Adherence times of
    leukocytes to vessel walls were log normally distributed, median values
    being 30, 130, and 560 sec, for lymphocytes, PMNs, and macrophages,
    respectively. Mean speeds of migration along luminal surfaces were similar
    (7-19 microns/min) for all three types of cells. Lymphocyte migration
    outward through the venular wall, observed on two occasions, took 1-2 min.
    Median values for duration of adherence of RBCs and lymphocytes to
    macrophages were 1 and 42 sec, respectively. Phagocytosis of a lymphocyte
    was observed and took 3 min. Macrophages often underwent dramatic changes
    in shape, including formation of a pseudopod up to 155 microns in length.

Schmidt EE; MacDonald IC; Groom AC.
     Comparative aspects of splenic microcirculatory pathways in mammals: the
     region bordering the white pulp.
   Scanning Microscopy, 1993 Jun, 7(2):613-28.
     (UI:  94151620)

    Splenic microcorrosion casts prepared using minimal volumes of
    material show that most of the flow passes through the region bordering the
    white pulp......... Capillaries never terminate within the reticulum
    of the white pulp. In immune thrombocytopenia, marked vascular hyperplasia
    occurs within white pulp and MZ.
    In ... spleens, the MS, MZ and PMCS are drained by open-ended venous 
    sinuses. ........ Approximately 90% of the splenic
    inflow passes through the region bordering the white pulp, bypassing the
    filtration beds of the red pulp.

Pellas TC; Weiss L.
     Deep splenic lymphatic vessels in the mouse: a route of splenic exit for
     recirculating lymphocytes.
   American Journal of Anatomy, 1990 Apr, 187(4):347-54.
     (UI:  90282186)

Abstract: A study of pathways of lymphocyte migration through mouse spleen
    revealed lymphatic channels closely following arteries in trabeculae and
    white pulp. Because there is no detailed record of the layout of deep
    splenic lymphatics in the mouse, or other species, we present our
    observations in this paper, relating our findings to normal migratory
    pathways of lymphocytes through the spleen. Lymphatics draining the spleen
    are so inconspicuous that they often are not mentioned in anatomical
    discussions. The data presented clearly demonstrate 1) the existence and
    layout of deep lymphatic vessels in the mouse spleen, and 2) that migrating
    lymphocytes exit white pulp via these lymphatic vessels. CD4+ and CD8+ T
    cell subsets migrated proximally along the central artery from distal
    (dPALS) to proximal periarterial lymphatic sheaths (pPALS) and exited via
    deep lymphatic vessels that originate there. B cells migrated from dPALS to
    enter lymphatic nodules (NOD), thus segregated from T cells. B cells then
    migrated toward and exited via deep lymphatics. The appearance of labelled
    lymphocytes in lymph coincided with their disappearance from white pulp
    compartments. Labelled T cells were observed in splenic lymphatics as early
    as 1 hr after intravenous infusion but took, on average, about 6 hr. B
    cells took somewhat longer. Thus T and B cells entered and left white pulp
    through shared pathways, but took divergent intermediate routes through
    dedicated zones, pPALS for T cells, NOD for B cells.
"Nothing more is needed to destroy a man, than the conviction that his
life's work is useless."  -Antonin Artaud

awarnock at cmgm.stanford.edu (R. Aaron Warnock)

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