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IMMUNOBIOLOGY
From the Departments of Molecular and Human Genetics,
Pediatrics, and Pathology, Baylor College of Medicine, Houston, TX; the
Department of Biomedical Engineering, University of Virginia Health
Sciences Center, Charlottesville, VA; and the Department of Comparative
Medicine, University of Alabama-Birmingham, Birmingham, AL.
In the initial phase of an inflammatory response, leukocytes
marginate and roll along the endothelial surface as a result of
adhesive interactions between molecules on the endothelial cells and
leukocytes. To evaluate the role of the 3 selectins (E, L, and P) in
leukocyte rolling and emigration, a null mutation for L-selectin was
introduced into previously described embryonic stem cells with null
mutations in the genes for both E-selectin and P-selectin (E/P
double mutants) to produce triple-selectin-null mice (E-selectin,
L-selectin, and P-selectin [E/L/P] triple mutants). Triple-selectin homozygous mutant mice are viable and fertile and only
rarely develop the severe mucocutaneous infections or pulmonary
inflammation characteristic of E/P double-mutant mice. Surface
expression of L-selectin was undetectable in triple-mutant mice on
fluorescence-activated cell-sorter analysis of peripheral neutrophils.
Pathological studies revealed moderate cervical lymphadenopathy and
lymphoplasmacytic infiltrate, but these were less extensive than in E/P
double-mutant mice. Neutrophil emigration during thioglycolate-induced peritonitis was significantly reduced at 4, 8, and 24 hours (35%, 65%, and 46% of wild-type values, respectively). Intravital
microscopy of the cremaster muscle revealed almost no rolling at times
up to 6 hours after exteriorization, with or without addition of tumor
necrosis factor Leukocyte emigration into the tissues after an
inflammatory stimulus is a process consisting of several distinct
steps. These have been described as (1) leukocyte rolling along
activated endothelium, (2) leukocyte activation, (3) firm adhesion to
the endothelium, and (4) transendothelial emigration.1,2
The selectins (E, L, and P) and their ligands are primarily responsible
for the initial leukocyte tethering and rolling events, and each
selectin has been shown to support rolling in vitro and in
vivo.3-9 L-selectin is expressed constitutively on most
leukocytes. E-selectin is expressed on endothelium, and up-regulation
requires de novo synthesis. P-selectin is expressed on endothelium and
platelets, and up-regulation occurs by rapid mobilization from
Mice with null mutations in the individual selectin genes have
been described previously. P-selectin-deficient mice10
and L-selectin-deficient4 mice both have defects in
leukocyte rolling, and neutrophil emigration into the peritoneal cavity
is delayed during thioglycollate-induced peritonitis.
L-selectin-deficient mice also have severe defects in lymphocyte
homing to the lymphoid tissues. E-selectin-null mice, though
originally thought to have no defects in the inflammatory
response,11 have been found to have defects in slow
leukocyte rolling12 and have increased mortality compared
with wild-type mice after intraperitoneal infection with
Streptococcus pneumoniae.13 All 3 of the
single-selectin-deficient strains of mice remain healthy under
specific-pathogen-free (SPF) conditions. The differences in the
phenotypes of individual-selectin knockout mice suggest distinct roles
for the selectins in inflammatory processes.
E-selectin and P-selectin (E/P) double-mutant mice have been described
by 2 research groups.10,14 These mice have profound leukocytosis, elevated levels of inflammatory cytokines,
hypergammaglobinemia, and severe defects in neutrophil emigration
during both chemically and bacterially induced
peritonitis.10,14 The mice also develop and eventually die
of spontaneous mucocutaneous infections. No leukocyte rolling was
observed in these mice after trauma or short-term treatment with tumor
necrosis factor Generation of mice that mimic an E-selectin, L-selectin, and P-selectin
(E/L/P) triple-mutant phenotype by transplantation of bone marrow from
L-selectin-deficient mice into irradiated E/P double
mutants17 showed a severe defect in leukocyte rolling in
these pseudo-triple-selectin-null mice. These observations were
confirmed when Robinson et al18 generated triple-selectin knockout mice genetically. Studies of these mice indicated a dominant role for P-selectin in leukocyte recruitment from the vasculature. The
mice also had severe defects in leukocyte emigration.
To further investigate the roles of the selectin molecules in vivo, we
independently generated mice deficient in all 3 selectin molecules.
These mice had defects in leukocyte rolling and emigration at least as
severe as those in double-selectin mutants, but surprisingly, they
appeared to be healthier, with a near absence of mucocutaneous and
pulmonary disease and greatly reduced leukocytosis compared with E/P
double-mutant mice.
L-selectin-gene targeting in embryonic stem cells
A scheme of negative selection followed by Southern blotting was used
to identify clones in which recombination had occurred in
cis with the previous mutations. Clones were selected for
loss of the chromosome containing the targeted E-selectin locus by using 6-thioguanine to select against the hypoxanthine
phosphoribosyltransferase (HPRT) cassette in the E-selectin gene. DNA
was prepared from the colonies surviving the selection, digested with
XbaI, and probed for concurrent loss of the L-selectin
mutation by Southern blot analysis. Clones that had lost both the
E-selectin and L-selectin mutations were judged to have the mutations
in cis. Injection of 2 clones into C57BL/6 embryos resulted
in germline transmission of the chromosome carrying the 3 mutations,
and no differences were observed for the 2 clones. The mice used in the
experiments were of a mixed C57BL/6 and 129/SvEv background and were
housed in a SPF barrier facility.
The mice prepared by our laboratory and those described by Hynes and
colleagues are referred to in the discussion section of this paper as
the Baylor (Bay) and Hynes (Hyn) mutations, respectively, in agreement
with the nomenclature of the Induced Mutant Resource of the Jackson
Laboratory
(http://jaxmice.jax.org/html/infosearch/searchDB_index.html). The
technical nomenclature for the genotypes so designated is as
follows:
Pathological assessments and blood counts
Blood was obtained from 8-week-old mice by means of bleeding of the retro-orbital venous plexus, collected in heparin-coated capillary tubes, and transferred to heparin-coated plastic tubes. All mice appeared healthy on gross examination at the time of complete blood counts done by using an automatic cell counter (Beckman Coulter, Fullerton, CA). Differential counts were performed in smears stained with Wright stain. Ten wild-type, 10 E/L/P triple-mutant, and 5 E/P double-mutant male mice were analyzed. Intravital microscopy Mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (Ketalar [100 mg/kg]; Parke-Davis, Morris Plains, NJ) after pretreatment with sodium phenobarbital (Nembutal [30 mg/kg given intraperitoneally]; Abbott Laboratories, North Chicago, IL) and atropine (0.1 mg/kg given intraperitoneally; Elkins-Sinn, Cherry Hill, NJ). A tube was inserted into the trachea and one jugular vein was cannulated for administration of anesthetic throughout the intravital microscopical experiment. One carotid artery was cannulated for blood pressure monitoring, blood samples, and systemic injections of monoclonal antibodies (mAbs). Mice were kept at a constant temperature of 37°C with use of a thermo-controlled heating lamp (Physitemp, Clifton, NJ) and received diluted phenobarbital in saline (0.2 mL/hour) intravenously to maintain anesthesia and a neutral fluid balance.The cremaster muscle was prepared for intravital microscopy as
described previously20 and was superfused with
thermo-controlled (35°C) bicarbonate-buffered saline. Blood samples
(10 ul each) were obtained from the carotid catheter throughout the
experiment at approximately 45-minute intervals to analyze systemic
leukocyte concentrations. Differential leukocyte counts were obtained
by evaluating blood samples stained with Kimura stain in a
hemocytometer. Microscopical observations were made by using a Zeiss
intravital microscope (Axioskop, Thornwood, NY) with a saline immersion
objective (SW 40/0.75 numerical aperture). Venules with diameters
between 20 and 80 fm were observed and the results recorded by using a CCD camera system (VE-1000CD; Dage-MTI, Michigan City, IN) on a
Panasonic S-VHS recorder. Center-line red blood cell velocity was
measured with a dual photodiode and a digital on-line cross-correlation program.21 Center-line velocities were converted to mean
blood flow velocities by multiplying by an empirical factor of
0.625.22 Wall shear rates ( Rolling and adhesion variables were determined as follows. Each
rolling leukocyte passing a line perpendicular to the vessel axis was
counted, and leukocyte rolling flux was expressed as leukocytes per
minute. Rolling flux fraction was calculated as described
previously20 by dividing leukocyte rolling flux by total
leukocyte flux estimated as white blood cell count (WBC) vb
Cytokine measurements and flow cytometry Enzyme-linked immunosorbent assays (ELISAs) for granulocyte-monocyte colony-stimulating factor (GM-CSF) and TNF-
were done according to the instructions of the manufacturer (R & D
Systems, Minneapolis, MN). Expression of L-selectin on leukocyte
populations was determined by flow cytometry analysis in 8-week-old
mice. Whole blood was collected and incubated with Fc Block (1:50;
Pharmingen, San Diego, CA) on ice for 5 minutes. Fluorescein
isothiocyanate (FITC)-labeled mAb to L-selectin (MEL-14 [1:50];
Pharmingen) or FITC-labeled isotope control was added, and the mixture
was incubated for 30 minutes. The granulocyte-specific,
isotope-matched, phycoerythrin-labeled mAb to Ly-6G (Gr-1; Pharmingen)
was used to identify the granulocyte population in the experiments
shown in Figure 2. After centrifugation and red blood cell lysis, flow cytometric analysis of L-selectin expression was done by using a Coulter Electronics XL-MCL flow cytometer (Beckman Coulter).
Peritonitis One milliliter of 3% thioglycolate (Difco, Mountain View, CA) was injected into the peritoneal cavity to induce peritonitis. At 4, 8, and 24 hours after injection, the mice were killed and peritoneal cells were collected by injecting 5 mL phosphate-buffered saline containing 0.1% bovine serum albumin, 0.54 mM EDTA, and 10 U/mL heparin (Sigma Chemical, St Louis, MO) and removing the peritoneal lavage. The total number of cells collected was determined with a hemocytometer. Cytospin preparations of the lavage were neat stained (Fisher Scientific, Pittsburgh, PA), and differential counts were used to determine the total number of neutrophils (differential percentage times total cells). For the peritonitis experiments with blocking of4-integrin, 50 µg mAb PS/2 was injected into the tail
vein 1 hour before and 6 hours after thioglycolate injection. Lavage
was collected and neutrophils were counted as described above.
Targeting of L-selectin in ES cells Triple-selectin-null mice were generated by targeting the L-selectin gene in ES cells containing previously introduced cis mutations in the genes for E-selectin and P-selectin.10 Because the 3 selectin genes in mice are closely linked on chromosome 1, triple-mutant mice could not be obtained readily by breeding of single-L-selectin- null mice with E/P double-selectin-null mice. The targeting construct (LSCON2-2) was designed to replace most of exon 3 with a puromycin resistance cassette by homologous recombination (Figure 1A). Exon 3 codes for the lectin-binding domain of L-selectin, and a similar deletion was used to produce a single- L-selectin-null mouse previously.4 The linearized construct was electroporated into double-mutant ES cells and selected for puromycin resistance. Genomic DNA from the surviving colonies was digested with XbaI, and Southern blotting with the 3' probe identified several clones with the desired 4.4-kb fragment expected for a replacement mutation. Further analysis using BglII and the 5' probe confirmed homologous integration of the construct, with a frequency of 1 in 30 of the puromycin-resistant colonies.A selection scheme was used to identify clones that had the integration event in cis or trans orientation to the 2 previous mutations. Cultures of individual clones were selected in 6-thioguanine to obtain subclones that had lost the chromosome with the HPRT cassette in the E-selectin locus. Genomic DNA from clones that had lost the E-selectin mutation was analyzed for the concurrent loss of the L-selectin mutation by Southern blotting. Loss of both the E-selectin and L-selectin mutations indicated that the integration had occurred in cis to the previous mutations (Figure 1B), resulting in cosegregation of the mutations that would generate the triple-null mouse when homozygous. Clones containing the triple-selectin mutation and the single-L-selectin mutation were injected into C57BL/6 blastocysts. This resulted in a high percentage of male chimeras that transmitted the mutations to the mouse germline. Intercrossing the progeny produced homozygous mice for both genotypes: triple-selectin-null mice and single-L-selectin-null mice. Southern blot analysis of tail DNA from the pups obtained from the mating of heterozygous triple-null mice demonstrated cosegregation of the mutations and generation of triple-null mice (Figure 1C). No evidence of prenatal or early postnatal death was found in either the triple-null or L-selectin-null mice, thereby confirming that the selectin genes are not essential for development or survival after birth. Surprisingly, the triple-null mice had less skin disease than the E/P double-null mice. Verification of null alleles Verification that the P-selectin and E-selectin mutations produce null alleles was reported previously.10,15 Eight-week-old wild-type (+/+) and homozygous null ( /) littermates from both the triple- and single-L-selectin mutants were tested for surface expression of L-selectin. Leukocytes from peripheral blood and bone
marrow of 8-week-old mice were incubated with FITC-labeled MEL-14 mAb
and analyzed by flow cytometry. Although wild-type mice had a
pronounced shift in fluorescence compared with the background level,
granulocytes from the triple-null mice did not show increased
fluorescence (Figure 2). This observation was made with all leukocyte
populations present in peripheral blood and bone marrow from both the
E/L/P triple-mutant mice and the single-L-selectin / mutant mice
(data for single mutant not shown).
White blood cell counts As shown in Table 1, introduction of the L-selectin mutation into E/P double-mutant mice resulted in partial reduction of the severe leukocytosis observed in double-null mice. The severe leukocytosis in the E/P double-mutant mice was consistent with previous observations in E/P double mutants generated from the ES cells used to make these triple-mutant mice10 and in other previously described E/P double-mutant mice.14 Leukocyte counts in triple-mutant mice were elevated over those in wild-type mice, including the counts of total WBCs (P = .0006), neutrophils (P < .0001), monocytes (P < .0001), and eosinophils (P < .05). There were no significant differences in lymphocyte counts between +/+ and triple-null mice (P = .519). However, addition of the L-selectin mutation resulted in a significant reduction in leukocytosis compared with findings in E/P double mutants. Total leukocyte counts were significantly lower in the E/L/P triple-mutant mice compared with the E/P double-mutant mice, including the counts of WBCs (P < .0001), neutrophils (P = .0097), lymphocytes (P = .0006), monocytes (P = .0002), and basophils (P = .0002). The reduction in leukocytosis observed when the L-selectin mutation was added to mice with mutations in E-selectin and P-selectin suggested decreased rather than increased inflammatory disease and was consistent with the decreased skin disease in the triple null mice.
Leukocyte rolling in triple null mice As expected, leukocyte rolling in the triple-selectin-null mice was severely compromised. Leukocyte rolling was completely absent at time points up to 5 hours after exteriorization of the cremaster muscle both with and without pretreatment with TNF-. Both rolling flux
(Figure 3A) and rolling flux fraction
(Figure 3B) were more reduced than in the E/P double-mutant mice,
again demonstrating the importance of L-selectin in rolling at a time long after the initial inflammatory stimulation (5 hours). The small
amount of rolling observed 5 hours after exteriorization was completely
eliminated by administration of mAb PS/2, which blocks the adhesive
function of 4-integrin (Figure 3); these results
indicate that this residual rolling is mediated by
4-integrin. The physiologic importance of this
4-integrin-mediated rolling is unclear.
Chemically induced peritonitis Neutrophil influx into the peritoneal cavity in the triple-null mice was compromised during thioglycolate-induced peritonitis. Significant inhibition of neutrophil emigration was present at all time points tested (Figure 4). The small amount of emigration observed indicated either that emigration may occur to some extent in the absence of rolling or that other molecules may act redundantly to mediate enough rolling in vivo for firm attachment followed by emigration. This was especially evident at the 24-hour time point, when more than 10 × 106 neutrophils were recovered from the peritoneal lavage. Because rolling mediated by4-integrin was revealed at later time points by
intravital microscopy, we hypothesized that blocking the action of
4-integrin might further reduce the number of emigrating
neutrophils, especially at the 24-hour time point. Figure 4 shows that
administration of mAb PS/2, which blocks function of
4-integrin, did not reduce neutrophil emigration at any
time point to a greater extent than did the deficiency of the 3 selectin molecules alone.
Cytokine assays ELISAs of 2 proinflammatory cytokines, GM-CSF and TNF-, were
done in wild-type mice, homozygous L-selectin mutant mice, E/P double-mutant mice, and E/L/P triple-mutant mice. GM-CSF levels were
increased in E/P double mutants compared with +/+ mice as expected
(Figure 5A) and reported
previously.14 Mice null for L-selectin had GM-CSF levels
similar to those in +/+ mice. Consistent with the decreased
leukocytosis in triple-null mice compared with E/P double-null mice,
levels of GM-CSF in triple-null mice were substantially lower than
those in E/P double-null mice and were not significantly different from
those in +/+ or L-selectin / mice (0.67 ± 0.46 pg/mL in E/L/P
triple mutants versus 0.34 ± 0.16 pg/mL and 0.37 ± 0.16 pg/mL in
+/+ and L-selectin / mice, respectively; P < .16 for
both). Introducing the L-selectin mutation into the E/P double mutants
prevented the 6- to 7-fold elevation of levels of GM-CSF observed in
the double mutants. Serum levels of TNF- in the E/L/P triple-mutant
mice were significantly higher than those in the +/+ or L-selectin
/ mice (Figure 5B). Although the difference was not statistically
significant (P = .1647), levels of circulating TNF-
were somewhat lower in the triple mutants compared with the double
mutants, similar to the findings for GM-CSF. The cytokine elevations
associated with the severe inflammatory disease of skin and lungs in
the E/P double mutants10,14 were reduced rather than
aggravated with introduction of a null mutation for the third
selectin.
Pathological findings As reported previously, mice null for E/P double mutations develop mucocutaneous infections or ulcerative dermatitis10,14 and progress to poor general health. Surprisingly, in our study, addition of L-selectin deficiency to the E/P null genotype virtually eliminated the development of skin disease and returned the mice to a much healthier appearance. Figure 6 shows 3 15-month-old littermate control mice (Figure 6A) and 3 E/L/P triple-mutant mice of the same age (Figure 6B); the mice were second filial generation C57BL/6 mice crossed with 129/SvEv mice. None of 34 triple-selectin-null mice that were housed in a SPF barrier facility for at least 15 months developed the severe skin disease observed at a much younger age in the E/P double mutants in the same mouse room.10,14 Only 3 of 156 triple-selectin-null mice (1.9%) had any dermatitis by the age of 6 months. These 3 mice (Figure 6C) were littermates, and the dermatitis observed was not as severe as that in the double-null mice. The E/L/P triple-mutant mice mated normally until at least 12 to 15 months of age and had much better overall health than the E/P double-null mice in the same mouse room, which only mated normally until 2 to 5 months of age.
On gross examination, cervical skin from the control,
L-selectin-deficient, and nearly all the E/L/P-selectin-deficient
mice was intact. In contrast, skin from the ventral portion of the neck
from most of the E/P double-mutant mice showed large areas of
ulceration. Representative histological sections of cervical skin from
these mice are shown in Figure 7.
Cervical skin from control, L-selectin-deficient, and
E/L/P-selectin-deficient mice (Figure 7A, 7B, and 7C, respectively)
had no excoriative lesions and was intact. Tissue sections of cervical
skin from these mice lacked a dermal infiltrate, and the surface had no
bacterial colonization. In contrast, histopathological examinations of
skin from E/P double-mutant mice (Figure 7D) revealed diffuse
excoriation and loss of the epidermis, with focal retention of the
basal cell layer of the epidermis. A mixture of gram-positive and
gram-negative bacteria colonized the surface of these denuded areas. An
intense mixture of granulocytes and mononuclear cells disrupted the
underlying pannicular muscle layer and infiltrated the underlying
dermis. No fungi or viral inclusions were observed. One of the E/L/P
triple-mutant mice (middle, Figure 6C) did have diffuse
ulceration similar to that in the E/P group. This mouse was one of the
3 of 156 triple-mutant mice mentioned above. However, less
granulocytic and mononuclear infiltration and less disruption of the
underlying dermis was observed in these mice (Figure 7E) than in the
E/P double-mutant mice.
On gross examination during necropsy, both the younger and older E/P-selectin-deficient and E/L/P-selectin-deficient mice had marked cervical lymphadenopathy, whereas L-selectin-deficient and control mice had cervical lymph nodes closer to normal in size. Control and L-selectin-deficient mice had relatively small and inconspicuous cervical lymph nodes, with preservation of typical nodal architecture in the wild-type mice, whereas the L-selectin- null mice had smaller nodes with disturbed architecture, as reported previously.4 Cervical lymphadenopathy was massive in E/P-selectin-deficient mice and was less extensive but still moderate to severe in E/L/P-selectin-deficient mice. In both the E/P double- and the E/L/P triple-mutant mice, the lymph nodes were replaced by a reactive lymphoplasmacytic infiltrate that effaced the typical corticomedullary architecture of the lymph nodes (data not shown). In the E/L/P triple-mutant mice, residual lymphoid follicles with germinal centers were observed occasionally, whereas these structures were not recognized in the E/P double mutants. The splenic tissue differed considerably among selectin-deficient and
control mice. Control mice had typical splenic architecture, with
well-defined lymphoid follicles with central arterioles separated distinctly by the sinusoids of the red pulp, which lacked
extramedullary hematopoiesis (Figure 8A).
The white pulp was relatively normal in spleens from
L-selectin-deficient mice, with only a mild degree of extramedullary
hematopoiesis in the sinusoids of the red pulp (Figure 8B). The
definition between red pulp and white pulp areas was not as distinct as
that in the normal spleens. Again, the E/P double mutants had the
greatest alteration in splenic parenchyma. In these mice (Figure 8C),
there was massive extramedullary hematopoiesis, with myeloid and
megakaryocytic lineages readily identifiable and a lesser erythroid
component. Lymphoid follicles forming the white pulp of the spleen were
effaced and observed infrequently because of expansion of the red pulp
by extensive extramedullary hematopoiesis. Spleens from younger and
older E/L/P-selectin-deficient mice had moderate disruption of the
splenic architecture by extramedullary hematopoiesis in the sinusoids
of the red pulp; however, lymphoid follicles of the white pulp were
identified occasionally and these had irregular sizes and shapes
(Figure 8D).
Lung tissue from the selectin-deficient mice had a gradient of
decreasing cellularity within the alveolocapillary walls, with the most
severely affected interstitial areas observed in the E/P double
mutants. Control (Figure 9A) and
L-selectin-deficient (Figure 9B) mice had no pulmonary alterations.
These groups of mice had lung parenchyma with typical architecture of
small airways and alveoli and lacked the bronchial lymphoid aggregates
observed in the younger and older E/L/P-selectin-deficient mice. The
12 alveolocapillary walls examined in the E/P double mutants were engorged by leukocytes to various degrees; however, these leukocytes were confined to the small vascular spaces and did not infiltrate the
interstitial supporting tissue or the alveolar and airway spaces
(Figure 9C). No organisms or viral inclusions were observed. There was
only a small increase in leukocytes in the alveolocapillary walls in
both the younger and older E/L/P-selectin-deficient mice, and
occasional small bronchial lymphoid aggregates were also present (Figure 9D). The appearances of alveolar and airway spaces in the lung
were moderately distorted but readily recognized. Again, no organisms
or viral inclusions were observed. Although there was some distortion
of lung architecture in the triple-null mice, it was never as dramatic
as that in the E/P double-mutant mice. Figure 9E shows a higher-power
magnification of lungs from an E/P double-mutant mouse; the cells
present are granulocytic.
We prepared mice with homozygous deficiency for all 3 selectin molecules. These E/L/P triple-null mutants had remarkably less inflammatory disease than we observed previously in E/P double-null mice. There are now 2 independently prepared mutant strains for E/L/P triple-null mice and E/P double-null mice: the Bay and Hyn mutations. The original reports on the E/PBay and E/PHyn mutants were similar in their descriptions of prominent skin disease, but they differed with respect to the lungs, which had no obvious abnormalities in E/PHyn mice but increased cellularity of the alveolocapillary walls in E/PBay mice.10 Although the findings in the lungs were not emphasized in the original report on E/PBay mice, they were usually quite prominent (Figure 9). There are also substantial similarities and some differences between the findings in the E/L/PBay mice reported here and those in observed E/L/PHyn mice.18 Both types of triple-null mice had leukocytosis, impaired neutrophil emigration, and a severe deficit in leukocyte rolling. The important differences were that E/L/PBay mice had minimal or no skin disease, whereas E/L/PHyn mice had skin disease as severe as that in E/PHyn double-mutant mice. The degree of leukocytosis correlated with the presence of severe skin disease; thus, leukocyte counts were lower in E/L/PBay triple-mutant mice than in the double mutants but higher in E/L/PHyn triple-mutant mice than in the double mutants. Because of the nature of the mutations introduced into the mice, it appears likely that all those mutations provide functional null alleles for all 3 selectins. It seems more likely that differences in observations are related to the genetic background of the mutant strains and differences in husbandry and environment. Exposure to opportunistic normal flora is likely to be an important variable in the development of skin disease, although extensive screening for pathogenic bacteria, fungi, and parasites yielded negative results in both mutant and sentinel mice. Organisms cultured from the severe infections in E/PBay mutant mice were opportunistic normal flora. Normal flora were also found in E/PHyn double-mutant mice.14 Although little or no skin disease was observed in the E/L/PBay triple-mutant mice during our experiments at Baylor or at the University of Virginia, a collaborator using these mice observed a higher incidence of skin infections in experiments conducted in Seattle (T. Papayannopoulou, personal communication, November 2000). Because our triple-mutant mice at another location had skin disease, we believe that the presence or absence of skin disease is related more to environment and microbiologic flora than to any difference between the E/L/PBay and E/L/PHyn mutations. It would be of interest to evaluate the effect of various microbes on the phenotypic differences with all mutations backcrossed onto a similar genetic background and all mutant mice kept in the same room. Results of studies of leukocyte rolling using intravital microscopy
were quite similar for the E/L/PBay and
E/L/PHyn triple-mutant mice and the E/PBay
double-mutant mice made additionally deficient for L-selectin by means
of bone marrow transplantation.17 In all 3 types of mice,
the rolling was more severely reduced than in mice with the double- or
single-selectin deficiencies. Rolling mediated by
The phenotype of the triple-selectin-null mice shows some similarity
to that of mice lacking the The amelioration of skin disease in the E/L/PBay mice compared with the E/PBay mice, although unexpected at the time of their generation, is not surprising when compared with the phenotypic improvement in E/PBay mice made additionally deficient for ICAM-1.26 Introduction of deficiency for ICAM-1 resulted in a complete rescue from the severe skin disease observed in the E/PBay double mutants, much like the amelioration observed in the E/L/PBay triple mutants. The types of skin lesions that occurred in most E/P double-mutant mice were not observed in 193 E/PBay mice that were also deficient for ICAM-1, including 14 mice older than 1 year. The amelioration in skin disease observed when a deficiency of either ICAM-1 or L-selectin was added to the E/PBay genotype may be explained by some functional similarities between ICAM-1 and L-selectin. Both L-selectin and ICAM-1 have roles in the development of T-cell responses and in emigration of neutrophils and other leukocytes. In specific immunity, ICAM-1 was found to be an important costimulatory molecule needed on splenic stimulator cells, as shown by the greatly diminished T-cell-stimulating activity in these cells in mice lacking ICAM-1.27 L-selectin is required for entry of lymphocytes into peripheral lymph nodes4 and for maintenance of normal lymphocyte recirculation through the body.28 It was also reported that mice deficient in L-selectin have impaired T-cell responses.29 Thus, both ICAM-1 and L-selectin have prominent roles in specific immunity. ICAM-1 and L-selectin have long been recognized as important components of the inflammatory response and leukocyte emigration.1,30 L-selectin is important in the early rolling of leukocytes along the endothelial surface at sites of inflammation, and ICAM-1 is important in firm adhesion of these same leukocytes. It was also shown that ICAM-1 and L-selectin have roles dependent on and overlapping each other for their proper function in leukocyte rolling and emigration.31,32 These observations are compatible with the finding that a deficiency of either ICAM-1 or L-selectin ameliorates skin disease in E/PBay mice. In our studies comparing E/PBay and E/L/PBay mice, we focused substantially greater attention on the prominent accumulation of leukocytes within the alveolar capillaries. Electron microscopy studies previously confirmed that this accumulation of leukocytes in the lung was retained within the vascular space.10 The pulmonary accumulation of leukocytes in the E/PBay mice but not the E/L/PBay triple-mutant mice may primarily reflect the substantially increased leukocytosis and some associated increase in proinflammatory cytokines. We support the interpretation that establishment (or not) of skin disease is the primary phenotypic variable, with changes in cytokine levels, blood count, and pulmonary leukocytes being secondary. Alternatively, the lung involvement may reflect a form of undetected infectious pneumonitis, but this seems unlikely given the extensive effort to identify microorganisms and the fact that leukocytes in the lung remain entirely within the vascular space. Microcirculatory obstruction in the lung with extreme hyperleukocytosis has been reported.33,34 There is a high frequency of respiratory distress in hyperleukocytic granulocytic leukemias, and similar pathophysiologic characteristics in E/PBay mice may provide a mouse model for this syndrome. In conclusion, there are important similarities and differences between
E/L/PBay and E/L/PHyn triple-mutant mice, as
well as marked differences between these mice and the corresponding E/P
double-mutant mice. In particular, severe compromise of the alveolar
space was observed in E/PBay mice but not in mice with any
other genotype. The severe skin disease and pulmonary disease in
E/PBay mice was greatly ameliorated by introduction of
deficiency of either L-selectin or ICAM-1 in addition to the E/P double
deficiency; this milder phenotype is similar to that observed in
Fuc-TVII
We thank D. Bulnes, P. Grennan, M. Idunoba, T. Mayer, F. Nails, S. Rodgers, J. Smalls, and E. Walker for histotechnology expertise. |
Top
Abstract
Introduction
2-integrins (eg, lymphocyte function antigen 1 and Mac-1).
w) were
estimated as 2.12 (8Vb/d), where Vb is the mean
blood flow velocity, d is the diameter of the vessel, and 2.12 is a
median empirical correction factor obtained from velocity profiles
determined by assessing microvessels in vivo.
(d/2),
