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The Journal of Immunology, 2008, 180, 2196 -2203
Copyright © 2008 by The American Association of Immunologists, Inc.
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Differential Molecular and Anatomical Basis for B Cell Migration into the Peritoneal Cavity and Omental Milky Spots1

Simon Berberich*, Sabrina Dähne*, Angela Schippers{dagger}, Thorsten Peters{ddagger}, Werner Müller{dagger}, Elisabeth Kremmer§, Reinhold Förster* and Oliver Pabst2,*

* Institute of Immunology, Hannover Medical School, Hannover, Germany; {dagger} Department of Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany; {ddagger} Department of Dermatology and Allergology, University of Ulm, Ulm, Germany; and § Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that {alpha}4β7 integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, {alpha}4β1 integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antibody production by adaptive immune responses requires several days for the activation, clonal expansion, and differentiation of conventional B2 cells and T cells. This vulnerable gap in the humoral response is bridged by the rapid production of natural Abs by specialized populations of B cells, including B1 cells. B1 cells are distinguished from conventional B2 cells by distinct functional properties, differentiation, phenotypes, and tissue distribution (1, 2, 3, 4). In particular, the body cavities, which are the peritoneal and pleural cavity, contain a prominent population of B1 cells.

Besides B1 cells the peritoneal cavity (PerC) harbors a prominent population of B2 cells. Indeed, B2 cells constitute the major B cell population in the PerC of most mouse strains and represent the sole B cell subset in the body cavities of adult humans. However, the functional properties of these peritoneal B2 cells have hardly been investigated so far. Peritoneal B2 cells share some features of B1 cells in that they acquire B1 cell characteristics in response to the local environment such as expression of CD11b and the ability to produce natural IgM (5). Moreover, we have recently shown that the PerC affects the migratory properties of B2 cells: Splenic B2 cells that have been exposed to the peritoneal environment re-entered this compartment more efficiently after adoptive i.v. transfer compared with unmanipulated splenic B cells (6). Such enhanced propensity to re-enter the PerC after exposure to stimuli present in that compartment is accompanied by modulation of the chemokine receptors CXCR4 and CXCR5 and integrins. CXCR5 is instrumental for B1 as well as B2 cell entry into the PerC (6, 7, 8), whereas the importance of other molecules involved in this process has not been addressed so far.

As a port of entry for B cells into the PerC the omentum has been proposed (7). The omentum is a bilayered sheet of mesothelial cells connecting various organs including spleen (SPL) and stomach. Inside the omentum, multiple B cell follicles are present that, based on their white color, have been termed milky spots. Consistent with a function of the omentum in B cell homing into the PerC, CXCL13 is highly expressed in the milky spots (9) and CXCL13-CXCR5 signaling is essentially required for the migration of adoptively i.v. transferred B cells into the PerC (6, 7, 10). Moreover, in short-term i.v. homing experiments, B cell accumulation in the omentum precedes appearance of the cells in the PerC (7). Similarly, accumulation of B cells in the omentum accompanies exit of B cells from the PerC after LPS stimulation (9). However, no direct experimental evidence supporting a role for the omentum in either B cell entry or B cell egress has been reported so far. With regard to B cell egress, lymphatic drainage into the parathymic lymph nodes (PTLN) has been suggested as an alternative pathway for B cell exit from the PerC.

In this study, we report that the entry of B2 cells into omental milky spots and the PerC requires different sets of integrins, indicating that entry into both compartments is independent of each other. Exit of peritoneal B2 cells requires the down-modulation of {alpha}4 integrins that initiates increased appearance of B2 cells in the PTLN. Surgical removal of the omentum does not result in a detectable failure of B2 cells to exit from the PerC, indicating that the omentum cannot be attributed a bottleneck function for B2 cells passing the PerC. Omental milky spots rather possess features of a secondary lymphoid structure, representing one of the available gateways for B2 cell circulation into and out of the PerC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 and C57BL/6 expressing the EGFP3 protein under control of the β-actin promoter (11) were bred in the Central Animal Facility of Hannover Medical School under specified pathogen-free conditions or were purchased from Charles River Laboratories. β2 integrin-deficient mice (C57BL/6 background) were a gift from K. Scharffetter-Kochanek (University of Ulm, Ulm, Germany) (12). β7 integrin-deficient (C57BL/6 background) (13), ICAM-1-deficient (C57BL/6 background) (14), mucosal addressin cell adhesion molecule 1 (MAdCAM-1)-deficient (C57BL/6 background), and MAdCAM-1/VCAM-1 double-deficient mice were bred at the Helmholtz Centre for Infection Research (Braunschweig, Germany). MAdCAM-1-deficient mice were generated in the laboratory of Dr. W. Müller and will be reported elsewhere. VCAM-1-deficient mice were generated by crossing mice homozygous for a loxP-flanked (floxed) Vcam1 gene (15, 16) and Mx-Cre-transgenic mice (17), resulting in a mouse in which Cre-loxP-mediated Vcam1 deletion can be induced by IFN injection. MAdCAM-1/VCAM-1 double-deficient mice on a mixed C57BL/6 x 129 Sv/Ev background were generated by crossing the MAdCAM-1-deficient mouse strain and the floxed VCAM-1 mouse. In the animals used for this study, deletion of the Vcam1 gene was induced by i.p. injection of 106 U of IFN-{alpha} 1 day after birth. Cre-negative littermates served as a control group. All mice were used at the age of 8–12 wk. All animal experiments have been performed in accordance with institutional guidelines and have been approved by the local government.

Bone marrow chimeras

Bone marrow cells were isolated from C57BL/6 mice expressing the EGFP protein under control of the chicken β-actin promoter and purified by discontinuous Lympholyte M gradient centrifugation. C57BL/6 wild-type recipients were lethally irradiated with a single dose of 10 Gy and reconstituted by i.v. injection of 107 syngenic bone marrow cells. Animals were analyzed 7–9 wk after reconstitution using a fluorescence stereomicroscope (Leica MZ 16FA; Leica) and Leica Application Suite software.

Antibodies

The following Abs were used: anti-β2 integrin-FITC, anti-CD23-PE (Caltag Laboratories), anti-{alpha}4β7 integrin, anti-β1 integrin-FITC, anti-β7 integrin, anti-CD19-allophycocyanin, anti-CD21/CD35-FITC, anti-CD31-FITC, anti-CD43-FITC, anti-CD62L-allophycocyanin, anti-MAdCAM-1, and anti-MECA-79 (BD Biosciences). Anti-B220 (clone TIB146) was conjugated to Cy5 or biotin as recommended by the manufacturer (Amersham Biosciences). Anti-{alpha}4 integrin (clone PS/2) and anti-L-selectin (clone MEL-14) Ab was raised under standard conditions and purified by affinity chromatography. Unconjugated primary Abs were detected using Cy5- or Cy3-conjugated mouse anti-rat Igs (Jackson ImmunoResearch Laboratories). Biotinylated Abs were recognized by streptavidin coupled to PerCP (BD Biosciences).

Cell isolation and flow cytometry

Mice were sacrificed by CO2 inhalation, and lymphoid organs were dissected and used for preparing single-cell suspensions by mincing through a 45-µm nylon mesh. Erythrocytes were lysed when necessary using a buffer containing ammonium chloride (1.7 M), potassium hydrogen carbonate (100 mM), and EDTA (1 mM). For isolation of peritoneal cells, the PerC was flushed with 10 ml of ice-cold PBS/3% FCS. Omental cells were isolated after incubation for 45 min with 1.5 mg/ml collagenase and 0.5 U/ml dispase (Roche) and mincing through nylon mesh. Cells were washed twice with PBS/3% FCS and stained with the Abs described above. Dead cells were excluded from further analysis by gating on DAPI– cells. Analysis was performed using a LSRII (BD Biosciences). Data were analyzed using WinList5.0 (Verity Software House) or FlowJo (Tree Star) software.

Whole mount microscopy

Omenta were dissected and fixed in 1% paraformaldehyde for 2 h, blocked with 5% of mouse or rat serum for 30 min, and incubated with anti-CD31-FITC, anti-B220-Cy5, and unconjugated anti-MAdCAM-1 or anti-MECA-79 Abs for at least 6 h. Specimens were washed twice for 30 min and, when appropriate, unconjugated Abs detected by Cy3-conjugated mouse anti-rat Ig. Omenta were washed twice for 30 min, refixed with 2% paraformaldehyde for 30 min, and mounted with Mowiol (Sigma-Aldrich). The whole staining protocol was performed at 4°C under continuous shaking. Slides were analyzed with a confocal laser scanning microscope (LSM 510 META; Zeiss). Images were created using Zeiss LSM5 and Imaris software (Bitplane).

Adoptive transfers

Splenocytes were labeled with CFSE or TAMRA (Molecular Probes). In brief, 2 x 106 cells/ml were preincubated for 30 min in RPMI 1640 medium containing 25 mM HEPES at 37°C. CFSE or TAMRA was added to a final concentration of 0.1 µM (CFSE) or 15 µM (TAMRA) for 10 min, followed by washing the cells in ice-cold PBS containing 3% FCS. For competitive adoptive transfer experiments, cell suspensions were adjusted to equal numbers of B cells labeled with each fluorochrome. Cell suspensions (107 cells/recipient) were injected i.p. and/or i.v. into the lateral tail vein of 8- to 12-wk-old syngenic recipients. Selective effects of the fluorescent staining procedure on the migration of cells were excluded by using both combinations of CFSE- and TAMRA-labeled cells throughout.

For the in vivo neutralization of integrins and L-selectin, TAMRA-labeled splenocytes were incubated with purified anti-{alpha}4 integrin (isotype: IgG2b, clone PS/2), anti-{alpha}4β7 integrin (isotype: IgG2a, clone DATK32), or anti-L-selectin Ab (isotype: IgG2a, clone MEL-14) for 15 min at room temperature. In brief, 107 cells were injected along with the Abs (anti-{alpha}4 integrin: 325 µg/mouse; anti-{alpha}4β7 integrin: 100 µg/mouse; anti-L-selectin: 100 µg/mouse) into wild-type recipients. As a control, cells were incubated with non-neutralizing isotype control Abs (IgG2a: clone 5D1D; IgG2b: clone 2B7-1).

In some experiments, 105 TAMRA-labeled splenocytes were injected along with 10 µg of LPS (Sigma-Aldrich) i.p. into wild-type recipients and omenta were analyzed 2 h after transfer using confocal microscopy (see above).

Explantation of omentum

For the surgical removal of the omentum, mice were anesthetized using a mixture of ketamine and rompun. A 1-cm longitudinal incision was made in the skin and a small opening was made in the upper left quadrant of the abdomen (laparotomy). The omentum was dissected where it attaches to the most distal part of the stomach (curvatura major). Sham operation consisted of abdominal incision and gentle manipulation of the omenta. The abdomen was closed in a single layer using discontinuous suture. Animals were used as adoptive transfer recipients 2 wk after surgery.

Statistics

Statistical analysis was performed on the original data with GraphPad Prism 4.0 software using the unpaired Student t test. Statistical differences for the mean values are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Integrin expression on peritoneal B cells and their function in peritoneal B cell homeostasis

Integrins are a group of heterodimeric adhesion molecules that are centrally involved in the homing of lymphocytes into lymphoid tissues. Integrin function on hemopoietic cells is mediated by β1, β2, and β7 integrins and the differential usage of these integrins critically contributes to the allocation of different immune cell subtypes to distinct immune compartments (18, 19). To investigate the function of integrins in the homeostasis of peritoneal B cells, we determined the expression level of integrins on peritoneal B1 and B2 cells and splenic B2 cells. β1, β2, β7, and {alpha}4 integrin were readily detected on peritoneal B1 cells as well as peritoneal and splenic B2 cells, although the different B cell subsets displayed divergent expression levels (Fig. 1A). Most evidently, peritoneal B2 cells expressed higher levels of β7 and {alpha}4 integrin and reduced expression of β2 integrin compared with splenic B2 cells (Fig. 1A). Integrin expression by peritoneal B1 cells is dominated by expression of β1 and {alpha}4 integrins that generally exceeds the expression levels observed on B2 cells (Fig. 1A). These results are consistent with a general role for integrins in regulating peritoneal B cell homeostasis. We next quantified B1 and B2 cells in the PerC of β2 and β7 integrin-deficient mice and in mice lacking the respective integrin ligands ICAM-1 and MAdCAM-1. Numbers of B1 and B2 cells were generally only mildly affected in these mutants and only the number of B2 cells in the PerC of ICAM-1-deficient mice was evidently increased compared with that of wild-type controls (Fig. 1B). Because MAdCAM-1 deficiency did not have any impact on the number of peritoneal B cells, we investigated the function of the β1 integrin ligand VCAM-1 for peritoneal B cell homeostasis in MAdCAM-1/VCAM-1 double-deficient mice. Because constitutive lack of VCAM-1 function is lethal, VCAM-1 was deleted postnatally by conditional gene deletion by Cre-recombinase induction. Interestingly, MAdCAM-1/VCAM-1 double-deficient mice but not MAdCAM-1 single-deficient controls displayed severely reduced numbers of peritoneal B1 and B2 cells (Fig. 1C), indicating that VCAM-1 might serve an essential function for homeostasis of peritoneal B cells.


Figure 1
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  		FIGURE 1. Integrins and their ligands contribute to B cell homeostasis within the peritoneal cavity. A, Expression of the indicated adhesion molecules was determined for peritoneal B1 cells (B220+CD23–), peritoneal B2 cells (B220+CD23+) as well as splenic B2 cells (B220+CD23highCD21int). Numbers indicate the ratio of the mean fluorescence intensity to the corresponding isotype control staining. Light gray shaded areas represent isotype controls for the cell populations indicated. B, The number of peritoneal B cells of 8- to 12-wk-old mice was analyzed for the different mouse strains indicated. B1 cells were addressed as CD19+CD43+ and B2 cells as CD19+CD43– cells. Bars depict the mean values (±SD) of total cell numbers (wild type: n = 30; β2 integrin-deficient mice: n = 8; β7 integrin-deficient mice: n = 10; MAdCAM-1-deficient mice: n = 7; ICAM-1-deficient mice: n = 5). C, The total number of peritoneal B1 and B2 cells was determined as in B. Cre-positive and Cre-negative littermates on a mixed C57BL/6 x 129Sv/Ev background were injected with IFN-{alpha} to initiate gene deletion 1 day after birth (see Materials and Methods). MAdCAM-1/VCAM-1/Crepositive: n = 14; MAdCAM-1/VCAM-1/ Crenegative: n = 6). The differences in the total number of peritoneal B cells isolated from MAdCAM-1-deficient mice displayed in B and C are likely to be caused by the different genetic backgrounds of the mice analyzed (C57BL/6 background in B and mixed C57BL/6 x 129Sv/Ev background in C).

 
β1 integrin but not β2 and β7 integrin mediates entry of B2 cells into the PerC

To directly explore the function of integrins for B2 cell entry into the PerC, we performed extensive adoptive transfer experiments. Splenocytes were isolated from wild-type and integrin-deficient mice and differentially labeled with fluorochromes. Mixtures of wild-type and integrin-deficient cells were adjusted to contain equal numbers of B cells and injected i.v. Sixteen to 17 h later, the homing efficiency of integrin-deficient cells into the SPL, mesenteric lymph nodes (MLN), peripheral lymph nodes, Peyer’s patches, blood, and the PerC was evaluated in comparison to wild-type cells. β2 integrin deficiency selectively impaired B cell homing into peripheral lymph nodes and Peyer’s patches, whereas β7 integrin deficiency reduced homing into Peyer’s patches and MLN (Fig. 2A, upper two panels). In contrast, none of both integrins affected the homing of B2 cells into the PerC. The redundancy inherent to the integrin/integrin ligand system may mask substantial contributions of distinct integrins for B cell homing. To rule out such compensatory effects, we next evaluated the homing of β2 integrin-deficient B2 cells in recipients lacking the archetypical {alpha}4β7 integrin ligand MAdCAM-1. In these experiments, we observed that homing of β2 integrin-deficient B2 cells into peripheral lymph nodes was further diminished in MAdCAM-1 recipients (Fig. 2, lower panel). Similarly, homing of B cells into MLN was almost completely abolished when β2 integrin-deficient B cells were transferred into MAdCAM-1-deficient recipients. These results indicate that β7 integrin-MAdCAM-1 interaction might compensate for residual homing of β2 integrin-deficient B2 cells in wild-type recipients and vice versa. Homing of β2 integrin-deficient B2 cells into the PerC was unaffected even in MAdCAM-1-deficient recipients, suggesting that β2 and β7 integrins might be dispensable for homing of B2 cells into that compartment.


Figure 2
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  		FIGURE 2. β2 integrin and β7 integrin are dispensable for B cell homing into the peritoneal cavity. The migration efficiency of β2 integrin-deficient or β7 integrin-deficient B cells in comparison to wild-type B cells was determined by competitive adoptive transfer experiments. Splenocytes were isolated from β2 integrin-deficient or β7 integrin-deficient as well as wild-type mice and differentially labeled with CFSE and TAMRA. Mixtures containing equal numbers of B cells were injected i.v. (A) or i.p. (B) into wild-type (A and B, upper two panels) or MAdCAM-1-deficient mice (A and B, lower panel). Sixteen to 17 h after adoptive transfer, the ratio of integrin-deficient to wild-type B cells was determined in different compartments by flow cytometry. Filled circles represent individual recipients and horizontal bars show mean values. PLN, Peripheral lymph node; PP, Peyer’s patches; n.d., not determined.

 
An analogous experimental setup was used to investigate the function of β2 and β7 integrins for B2 cell exit from the PerC following i.p. transfer of differentially labeled splenocytes. These experiments showed that β7 integrin is not involved in B2 cell exit from the PerC, whereas β2 integrin deficiency reduced the ability to leave the PerC 1.5-fold in wild-type as well as MAdCAM-1-deficient recipients (Fig. 2B).

Since neither β2 nor β7 integrin appeared to play a major role in the migration of B cells into the PerC, we extended our studies to {alpha}4 integrin and L-selectin. {alpha}4 integrin can associate with the β1 integrin chain to form {alpha}4β1 heterodimers (VLA-4) and the β7 integrin chain resulting in {alpha}4β7 dimers (LPAM-1). {alpha}4 integrin and L-selectin function was impaired in vivo by neutralizing Abs (see Materials and Methods). Wild-type splenocytes were fluorescently labeled, preincubated with the neutralizing Abs or the same amount of isotype control Abs, and constant cell numbers were transferred along with neutralizing amounts of Ab into wild-type recipients. Interestingly, in vivo neutralization of {alpha}4 integrin function but not L-selectin reduced the number of adoptively transferred B2 cells recovered from the PerC (Fig. 3), indicating that {alpha}4 integrin might mediate immigration of B2 cells into the PerC. Likewise, {alpha}4 integrin neutralization reduced the number of B2 cells recovered from the PerC after i.p. transfer (Fig. 3A, right panel), suggesting that {alpha}4 integrin is involved in the retention of cells in this compartment. Consistently, anti-{alpha}4 integrin neutralization increased the number of i.p. transferred cells recovered from lymphoid organs, further supporting a function for this integrin in retaining cells in the PerC. Because we did not observe any function for β7 integrin in homing into the PerC nor in cell egress from this site, these data implicate that {alpha}4β1 integrin is involved in both processes.


Figure 3
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  		FIGURE 3. In vivo neutralization of {alpha}4 integrins decreases B cell homing into the peritoneal cavity and induces B cell exit. The migration efficiency of wild-type splenocytes in the presence of neutralizing anti-{alpha}4 integrin (A), anti-L-selectin (B), or an isotype control Ab was determined in adoptive transfer experiments. Splenocytes were isolated from wild-type mice, labeled with TAMRA, preincubated with neutralizing Abs, and injected along with the Abs i.v. (left panel) or i.p. (right panel) into wild-type recipients. Sixteen to 17 h after adoptive transfer, the percentage of TAMRA+ B cells within different compartments was determined by flow cytometry. Data obtained in different experiments were normalized to the frequency of TAMRA+ B cells isolated from the different compartments of isotype control Ab-treated recipients. Filled circles represent the normalized migration efficiency observed in individual mice in the presence of neutralizing Abs (+) and isotype control Ab (–). PLN, peripheral lymph node.

 
{alpha}4β7 integrin mediates homing of B2 cells into omental milky spots

Migration of B cells into the PerC has been proposed to route through the omentum. The murine omentum is rich in B cell follicles, i.e., milky spots, and might contribute to homing of B cells into the PerC. However, experimental evidence for such function of the omentum is mostly indirect. We thus investigated the distribution of milky spots in vivo. To this end, wild-type C57BL/6 mice were lethally irradiated and reconstituted with syngenic bone marrow constitutively expressing the GFP. Eight weeks after reconstitution, bone marrow chimeras were sacrificed and the distribution of fluorescent cell aggregates in the omentum and other tissues was inspected using a fluorescent stereomicroscope. Fluorescent aggregates corresponding to milky spots were strictly confined to the omentum as morphologically separate tissues, whereas no such structures were present at other sites throughout the peritoneum (Fig. 4A).


Figure 4
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  		FIGURE 4. B cell entry into the omental milky spots requires β7 integrins. A, Localization of the omentum within the peritoneal cavity of a wild-type mouse (left panel). Milky spots were visualized in bone marrow chimeras expressing the EGFP protein in hemopoietic cells (right panel). Int, Small intestine; Om, omentum; Spl, spleen; Stm, stomach. B, Homing efficiency of i.v. transferred splenocytes into omental milky spots. Cells were isolated from wild-type and β7 integrin-deficient mice and labeled with TAMRA. Cell were transferred i.v. into either untreated wild-type recipients or into recipients injected with neutralizing Abs directed against {alpha}4 integrin, {alpha}4β7 integrin, and L-selectin or a non-neutralizing isotype control. Sixteen to 17 h after i.v. transfer, the number of transferred cells in milky spots (addressed as B220+ cell clusters) was determined by confocal microscopy on whole mount-stained omenta. As exemplified in the left panel, TAMRA+ cells (red) are readily detectable in omental milky spots defined as B220-expressing cell aggregates (blue), marked by a dashed white line. The number of TAMRA+ cells per area was normalized to the density of cells observed in isotype control Ab-treated recipients. Filled circles represent the density of adoptively transferred cells in the milky spots of individual mice. In the case of β7 integrin-deficient donor cells, one value ({circ}) was excluded from the analysis. C, Anti-MAdCAM-1 and anti-MECA-79 binding in omental milky spots of wild-type mice. Intact omenta were stained as whole mounts for B cells (anti-B220, blue), vessels (anti-CD31, green), and anti-MAdCAM-1 (left panel, red) or anti-MECA-79 Ab (right panel, red). Figures were generated using confocal microscopy. In each case, one representative milky spot is shown.

 
To investigate the homing of adoptively transferred cells into the omentum, we established a method that allows analyzing the number and distribution of cells in the omentum. To this end, cells in the omentum were stained in whole mounts, i.e., in the intact tissue. Subsequently, confocal microscopy was used to reconstruct the three-dimensional architecture of the tissue and to quantify the number of transferred TAMRA+ cells within individual omental milky spots (Fig. 4B, left panel). Using this technique, we determined the homing efficiency of β7 integrin-deficient B cells as well as wild-type B cells in the presence of neutralizing anti-{alpha}4 integrin, anti-{alpha}4β7 integrin, and anti-L-selectin Abs in vivo following i.v. transfer. Interestingly, genetic as well as Ab-mediated impairment of β7 integrin function inhibited the homing of B cells into omental milky spots (Fig. 4B, right panel). In contrast, inhibition of L-selectin did not decrease homing of B cells into the omentum but leads to an accumulation within that tissue. This effect might be attributed to the impaired B cell entry into peripheral lymph nodes following L-selectin blockade, resulting in overall increased numbers of B cells within the circulation.

Consistently, we observed that MAdCAM-1 is expressed by a subset of CD31-expressing vessels in the milky spots (Fig. 4C, left panel) but not in vessels outside the milky spot region. Likewise, we noticed binding of MECA-79 Ab recognizing sialy-LewisX glycosylation patterns on CD31-expressing vessels exclusively in milky spots (Fig. 4C, right panel). When analyzing large numbers of individual milky spots, we observed that MECA-79-binding structures were roughly twice more frequent compared with MAdCAM-1 Ab-binding vessels (20.9% MECA-79-positive vs 11.6% MAdCAM-1-positive structures among of all CD31-expressing vessels). However, both epitopes were recognized in almost every milky spot, indicating that MECA-79- as well as MAdCAM-1-binding vessels are a general hallmark of omentum milky spots. MECA-79 reactivity is a typical feature of high endothelial venules inside lymph nodes, where these particular vessels support transit of cells from the blood into the organ. Indeed, the staining pattern detected by MECA-79 Ab overlaps with that of MAdCAM-1, suggesting that MAdCAM-1-positive vessels represent the gateway for B cell immigrating from blood. Yet the presence of sialy-LewisX-positive vessels does not depend on MAdCAM-1 expression, because MECA-79 binding in wild-type and MAdCAM-1-deficient omenta was comparable (data not shown).

Peritoneal B cells enter omental milky spots

To further characterize the interrelation of B cells in the PerC and the omentum, we tracked the positioning of i.p. transferred cells in the omentum by three-dimensional reconstruction of confocal images. Fluorescently labeled, adoptively transferred splenocytes were abundantly scattered throughout the omentum 1 h after i.p. transfer without any obvious enrichment in either milky spots or along CD31-expressing vessels (Fig. 5A). Two hours after transfer, the cells started to aggregate in the region of omental milky spots and this process was virtually completed after 16 h. At this time point, the majority of all omentum-resident cells was confined to milky spots, with only a few cells scattered in the surrounding regions. Notably, at all times, virtually all transferred cells detected in the omentum expressed B220 (data not shown), suggesting that almost exclusively B cells rapidly enter milky spots from the PerC in this experimental setup. At all time points analyzed, adoptively transferred cells, by far exceeding in number the cells in the omentum, could be isolated by peritoneal lavage (data not shown). This indicates that only a minority of cells settled in the omentum, whereas the majority associated with other niches within the PerC. We therefore propose that the rapid entry of i.p. transferred cells into the omentum reflects a wave of freely "disposable" cells that did not yet adhere to local niches. In contrast, at later time points after transfer, most cells will have made stable contacts with local matrix, resulting in fewer cells entering the omentum despite overall high numbers of B cells in the PerC.


Figure 5
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  		FIGURE 5. Cell migration from the PerC into omental milky spots depends on the availability of mobile cells. A, Positioning of splenocytes at different time points after i.p. transfer. Splenocytes from wild-type mice were isolated, labeled with TAMRA, and 107 cells were injected i.p. into wild-type recipients. One, 2, and 16 h after transfer, recipient mice were sacrificed and the omenta were isolated. Whole mount stainings were performed using Abs against B220 (blue) and CD31 (green) and analyzed using confocal microscopy. Figures show distribution of TAMRA+ cells within omental milky spots 1 h (upper panel), 2 h (middle panel), or 16 h (lower panel) after transfer. B, Splenocytes from wild-type mice were isolated, labeled with TAMRA, and 8 x 106 cells were injected i.p. into wild-type recipients. Twenty-four hours later, the same recipients got another 8 x 106 splenocytes i.p. that were labeled with CFSE. Sixteen to 17 h later, recipient mice were sacrificed, and the omenta were isolated and analyzed using confocal microscopy. The figure shows distribution of transferred TAMRA+ or CFSE+ cells within one representative omental milky spot 2 days after the initial transfer. C, Mobilization of transferred cells into the omental milky spots following i.p. application of LPS. Splenocytes from wild-type mice were isolated, labeled with TAMRA, and 105 cells were injected along or without 10 µg of LPS i.p. into wild-type recipients. Two hours after transfer, omenta were isolated from the recipient mice and whole mount stainings were performed using Abs against B220 and CD31. Numbers of TAMRA+ splenocytes within omental milky spots (addressed as aggregates of B220+ cells) were determined using confocal microscopy. The number of TAMRA+ cells per mm2 omental milky spot was normalized as described for Fig. 4B. The diagram shows migration of transferred splenic cells with or without LPS treatment into the omental milky spots of wild-type mice (•). Black bars show mean migration of transferred splenic cells. D, The relative percentage of peritoneal B1 and B2 cells was determined 3 h after i.p. injection of 10 µg of LPS. B1 cells were addressed as CD19+CD3–CD43+ cells and B2 cells as CD19+CD3–CD43– cells. Filled circles represent the percentage of B1 and B2 cells within the total peritoneal B cell population of individual mice. Horizontal columns represent the mean percentage of the two peritoneal B cell subsets.

 
To test this hypothesis, we transferred differentially labeled splenocytes in two waves: TAMRA-labeled cells were i.p. transferred and allowed to settle in the PerC and omentum of wild-type recipients. Twenty-four hours later, CFSE-labeled cells were injected into the same recipients and the mice were sacrificed 1 day after the second transfer. Confocal microscopy of these recipients revealed that cells injected at both time points populated the omentum with the same kinetics and pattern as described for first-wave immigrants (Fig. 5B). This indicates that there is no space limitation, preventing peritoneal B cells from entering the omentum. Intraperitoneal injection of TAMRA-labeled splenocytes along with LPS into recipient mice boosted the number of B cells present in the omentum (Fig. 5C). Thus, activation of B cells appears to counteract settlement in the PerC and thereby might trigger sustained immigration into the omentum and egress from the PerC. Consistently, we noted that i.p. injection of LPS reduced the number of peritoneal B1 and B2 cells similarly, as evidenced by a constant peritoneal B1:B2 cell ratio in the presence of overall reduced B cell counts in the PerC (Fig. 5D).

Since B2 cell homing into the omentum after i.v. transfer depends on β7 integrin, we investigated whether this integrin would also mediate homing of B2 cells into the omentum after i.p. transfer. Quantifying the number of adoptively transferred cells in the recipients’ omentum 2 h after i.p. injection, we did not notice any difference in the frequency of wild-type and β7 integrin-deficient cells in the recipients’ omenta (data not shown). This shows that in contrast to the i.v. route the entry of cells into the omentum from the PerC does not depend on β7 integrin.

Surgical removal of the omentum reduces homing of i.v. transferred B cells into the PerC but does not impact accumulation of i.p. transferred cells in lymphoid organs

The divergent role of β7 integrin for B2 cell entry into the omentum and the PerC (cf Figs. 2A and 4B) demonstrates that B2 cell entry into the PerC does not necessarily route through the omentum. We next directly addressed the function of the omentum in B cell homing into the PerC through surgical removal of that tissue. As a control, a second group of mice received mock surgery. Two weeks after surgery, fluorescently labeled wild-type splenocytes were adoptively transferred i.v. and i.p. and the number of donor cells was determined 16–17 h later in various compartments. Interestingly, omentonectomy resulted in a 40% reduction of i.v. transferred cells migrating to the PerC compared with recipients that underwent mock surgery (Fig. 6A, left panel). In contrast, removal of the omentum did not affect the frequency of i.p. transferred cells in any of the organs analyzed (Fig. 6A, right panel). These data suggest that the omentum can contribute to the homing of splenocytes into the PerC, whereas it is not involved in mediating egress from this site. In contrast, i.p. transferred cells were strongly enriched in the PTLN draining the PerC compared with peripheral and MLN (Fig. 6B). This result indicates that egress of B cells from the PerC might mostly occur via draining lymphatic vessels.


Figure 6
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  		FIGURE 6. Removal of the omentum decreases B cell entry into the PerC but does not impact B cell exit from that compartment. A, Omenta of wild-type mice were surgically removed. Two weeks after explantation, 107 TAMRA-labeled splenocytes of wild-type donors were transferred i.v. (left panel) or i.p. (right panel) either into these animals or into recipients that were sham operated. Sixteen to 17 h after adoptive transfer, the percentage of TAMRA+ B cells within different compartments was analyzed using flow cytometry. B cells were addressed as B220+ cells showing typical characteristics of lymphocytes regarding their forward and sideward scatter. Data obtained in different experiments were normalized to the frequency of TAMRA+ B cells isolated from the different compartments of control recipients. Filled circles represent the normalized migration efficiency observed in individual mice after removal of the omentum compared with control mice. Black bars show mean migration of B cells into each compartment analyzed. Obvious statistical outliers ({circ}) were excluded from the calculation of mean values. ILN, Inguinal lymph node. B, B cells preferentially accumulate within the PTLN following i.p. transfer. In brief, 107 TAMRA-labeled splenocytes were transferred i.p. into wild-type recipients. Sixteen to 17 h after adoptive transfer, percentages of TAMRA+ B cells were determined within inguinal, mesenteric, and PTLN using flow cytometry. B cells were addressed as B220+ cells showing typical characteristics of lymphocytes regarding their forward and sideward scatter. Data obtained in different experiments were normalized to the frequency of TAMRA+ B cells isolated from inguinal lymph nodes. Filled circles represent the normalized frequency of TAMRA+ B cells observed in individual mice. Black bars show mean frequency of TAMRA+ B cells within different compartments analyzed compared with the frequency within the inguinal lymph nodes. ILN, Inguinal lymph node; ns, not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
B2 cells constitutively egress from the PerC entering the circulation and subsequently other lymphoid compartments such as the SPL. Similarly, B cells are able to enter the peritoneum from the circulation. In this study, we analyzed the molecular and anatomical basis for these migration processes. As summarized in Fig. 7, a divergent set of integrins mediates the migration of B2 cells from the circulation into omental milky spots and into the PerC. Migration of B2 cells from the circulation into omental milky spots requires {alpha}4β7 integrin, whereas {alpha}4β1 integrin mediates the direct entry of B2 cells into the PerC and their retention in the peritoneum (Fig. 7).


Figure 7
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  		FIGURE 7. Schematic presentation of B2 cell migration into the PerC and egress from this compartment under homeostatic conditions. B2 cells enter the PerC via two independent routes: 1) {alpha}4β1 integrin mediates direct migration of B2 from the circulation into the PerC and 2) blood-borne B2 cells enter omental milky spots by β7 integrin-MAdCAM-1 interaction and subsequently might transit into the PerC. Inside the PerC, the majority of B2 cells are retained in the compartment by {alpha}4 integrin-dependent adhesion to the local matrix. This interaction limits the pool of nonadherent peritoneal B2 cells that can exit this compartment in a β2 integrin-mediated mechanism via the draining lymph nodes or alternatively enter omental milky spots from the peritoneal side. Dashed lines indicate putative migratory routes that have not yet been experimentally shown.

 
Immigration of B cells into the PerC as well as egress from this site have been suggested to route through the omentum (7, 9). However, here we show that surgical removal of the omentum does neither abolish B cell homing into the PerC nor exit from this site. Thus, the omentum cannot be attributed a bottleneck function for B2 cells entering or exiting the PerC and alternative pathways leading B cells into and out of the PerC must exist.

Accumulation of B2 cells in milky spots can be observed already 1 h after adoptive i.v. transfer (this study and Ref. 7). Thus, B cells can be observed in milky spots before detectable numbers of B cells in the PerC. This indicates that B cell entry into milky spots is a more rapid process in comparison to B cell entry into the PerC. B2 cell entry into milky spots from the circulation does not depend on L-selectin (Fig. 4B), but is strictly dependent on CXCL13 (7) and, as shown in this study, on β7 integrin-MAdCAM-1 interaction. Consistently, CXCL13 is detectable in milky spots (9). Moreover, we observed that CXCL13 could be detected on the luminal side of MECA-79-binding venules in omental milky spots (data not shown). In addition, CD31-expressing vessels vascularizing the milky spots express MAdCAM-1 and resemble classical high endothelial venules by virtue of their MECA-79 stain (Fig. 4C). Thus, the molecular constituents enabling B cell entry into milky spots are similar to those of B cell transmigration into Peyer’s patches (20, 21).

Apart from entering milky spots from the circulation, B2 cells can also colonize this tissue from the peritoneal side. However, this process is rather scarce under constitutive conditions as most of the peritoneal B2 cells make stable contacts to the local matrix via the interaction of adhesion molecules with their ligands. Recently, mobilization of peritoneal B1 cells in response to i.p. injection of LPS has been shown. LPS triggered the down-modulation of {alpha}4 integrin and CD9 on peritoneal B1 cells, initiating massive egress from the PerC and accumulation of B1 cells in the omentum (9). In the current study, similar effects could be observed for B2 cells following i.p. injection of LPS or after i.p. transfer of B2 cells (Fig. 5). In both cases, numerous B cells accumulate within omental milky spots, indicating that B2 cells that do not make stable contacts to the local matrix are present in the PerC. Thus, migration of both, peritoneal B1 and B2 cells, into the omentum might be a result of freely available nonadherent B cells in the PerC. The molecular mechanisms and the rate of peritoneal B2 cell migration into milky spots are unknown. In contrast to B2 cell entry into milky spots from the circulation, β7 integrin is dispensable for B2 cell entry from the peritoneal side and one can only speculate on a putative role for CXCL13-CXCR5 interaction in leading B2 cells into milky spots.

In conclusion the omentum receives B2 cells from two different sources: 1) Blood-borne B2 cells enter milky spots via vessels resembling high endothelial venules and 2) peritoneal B2 cells apparently crawl throughout the tissue toward milky spots. The subset composition of omental B cells reflects these different influxes: B1 cells constitute 35.0 ± 7.7% of all peritoneal B cells but only 19.6 ± 8.8% (n = 11 mice analyzed) of all omentum-resident B cells and are hardly detectable in blood. Constitutive entry of B cells into milky spots needs to be balanced by corresponding numbers of B2 cells leaving the milky spots. Although we did not investigate this process, one might speculate that B2 cells exiting from milky spots might enter the circulation and/or the PerC. Therefore, the omentum might serve as a relay station for B cells present in the circulation and the PerC (Fig. 7), allowing transit of B cells from the circulation into the PerC. However, accumulation of i.v. transferred B2 cells in the PerC is drastically delayed compared with accumulation in milky spots, indicating that such a process appears to be counteracted by the exit of omental B2 cells back into the circulation. Thus, despite the fact that the omentum may enable entry of B2 cells from the circulation into the omentum, it certainly does not represent a highway for B2 cell migration and should rather be regarded as a separate lymphoid compartment.

Our results obtained by surgical removal of the omentum as well as the divergent role of β7 integrin in mediating entry of i.v. transferred B2 cells into the omentum but not into the PerC indicate the existence of alternative routes for B2 cell entry. Neutralizing anti-{alpha}4 integrin Ab reduces the immigration of splenocytes into the PerC. {alpha}4 integrin forms heterodimers with both the β1 and the β7 integrins. Because genetic ablation of β7 integrin does not impair homing of i.v. transferred B2 cells into the PerC, this observation argues for an essential role of {alpha}4β1 integrin in B cell migration into the PerC. {alpha}4β1 integrin interacts with VCAM-1 and indeed we observed reduced numbers of peritoneal B cells in MAdCAM-1/VCAM-1 double-deficient mice but not MAdCAM-1 single-deficient mice. The anatomical site for {alpha}4β1 integrin/VCAM-1-mediated B2 cell entry is unknown and one can only speculate that postcapillary venules expressing VCAM-1 might mediate this process. Notably, VCAM-1 is not expressed constitutively in the omentum (22), further supporting the idea that {alpha}4 integrin-mediated B2 cell entry from the circulation into the PerC does not route through this structure. {alpha}4β1 integrin also retains B2 cells in the PerC as revealed by reduced numbers of i.p. transferred B2 cells in the PerC in the presence of neutralizing anti-{alpha}4 integrin Ab. This effect might add to the reduced appearance of B cells in the PerC after i.v. transfer. However, it is unlikely to fully account for the observed effect, because both i.p. and i.v. transfers revealed a similar degree in reduction after anti-{alpha}4 integrin Ab treatment. We thus suggest that {alpha}4 integrin-mediated adhesion of peritoneal B2 cells to local niches might limit the availability of peritoneal B2 cells for exit as well as migration into the omentum. Therefore, modulation of {alpha}4 integrin function might constitute a critical regulatory element in peritoneal B2 cell homeostasis.

Egress of B2 cells from the PerC appears to route through the draining PTLN. Indeed, filtration of i.p. injected particulate material by PTLN is known for more than one century (23) and also accumulation of i.p. injected macrophages in these lymph nodes has been described already in 1970 (24, 25). Recently, accumulation of peritoneal B cells within the PTLN could be demonstrated in FTY720-treated mice. Because FTY720 is know to impair the egress of lymphocytes from lymph nodes, this finding indicates that B cell existing from the PerC pass the PTLN (26). Likewise, i.p. transferred B cells are more frequent in the PTLN compared with other nondraining lymph nodes (Fig. 6B). However, at present, we cannot rule out alternative pathways bypassing the PTLN.

In conclusion, our data reveal that divergent molecular mechanisms mediate entry of B2 cells into the PerC and into omental milky spots. Peritoneal and omental B2 cells exchange between both compartments but there is no linear relationship that would qualify the omentum as a major port of entry for B2 cells into the PerC. Instead, {alpha}4β1 integrin-mediated homing into the PerC and retention of B2 cells inside this compartment might represent a major mechanism controlling peritoneal B2 cell homeostasis.


   Acknowledgments
 
We thank Günter Bernhardt, Immo Prinz, and Andreas Krueger for critically reading this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Deutsche Forschungsgemeinschaft Grant SFB621-A1 (to R.F.) and Forschergruppe FOR 471/2 (to W.M.). S.D. was supported by the International Research Training Group 1273, funded by the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Oliver Pabst, Institute of Immunology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail address: Pabst.Oliver{at}MH-Hannover.de Back

3 Abbreviations used in this paper: EGFP, enhanced GFP; MAdCAM-1, mucosal addressin cell adhesion molecule 1; MLN, mesenteric lymph node; PerC, peritoneal cavity; SPL, spleen. Back

Received for publication September 4, 2007. Accepted for publication November 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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