Abstract Splenic marginal zones (MZ) are architecturally organized to generate a rapid response against blood borne antigens entering the spleen. Proper alignment includes MZ B cells, MARCO+ MZ macrophages (MZM), and MOMA1+ metallophilic macrophages positioned around a MAdCAM-1+ cell lined sinus. Our objective was to assess cell specific morphological changes of the MZ in young vs aged mice. We observed gross changes in the spleens of aged mice that reflect the improper alignment of cells in the MZ. Immunocytochemistry and immunofluorescence were used to reveal the positioning of MAdCAM-1+ sinus lining cells and MZM or MOMA1+ macrophages. As reported, in young mice, a continuous single cell layer of MAdCAM-1+ cells enveloped the white pulp, in addition to a cohesive line of MZM and MOMA1+ macrophages closely aligned with MAdCAM-1+ cells. In contrast, the MAdCAM-1+ cells of aged mice were more diffuse and less organized. MOMA1+ macrophages were also discontinuous and highly diffused into the white pulp and B-cell follicles. MAdCAM-1+ cells were surrounded by discontinuous, patchy groups of MZM. The reduction of MZM in aged spleens was confirmed by evaluating the ability of MZM to bind the FITC-labeled Dextran in vivo. By flow cytometry, the reduction in MZM in individual aged mice statistically correlated with reduced frequency of MZB cells. These changes may affect the ability of the MZ to properly respond to and clear pathogens in aged mice. NIH RO1AG013874 and T32AG031780
Abstract Alcohol intoxication is involved in 50% of all burn injuries, resulting in an increased risk of lung infection and failure, relative to burn alone. Our work has shown that in a mouse model of episodic binge ethanol exposure followed by burn injury there is prolonged pulmonary inflammation, relative to burn alone. The magnitude of pulmonary inflammation is mediated in part by alveolar macrophages (AMs), thus we examined the effect of ethanol on AM activation. Thirty minutes after binge ethanol exposure, mice were anesthetized and given a 15% total body surface area dorsal scald injury. At 24 and 72 hrs post-injury bronchoalveolar lavage AM granularity was assessed by flow cytometry using side scatter (SSC). At 24 hrs, 12% of AMs from burn alone had high SSC relative to 1.5% of AMs from sham groups (p<0.05). Only 6% of AMs from combined injured had high SSC. By 72 hrs, 3% of AMs from both burn groups had high SSC. Anti-inflammatory M2 phenotype of high SSC AMs was assessed using M2 marker CD206. At 24 hrs, there was no change in CD206. At 72 hrs, AMs from burn alone had a 43% increase in CD206 relative to sham groups. Giving ethanol prior to burn further elevated the expression of this marker by 46% over burn alone. These data indicate ethanol exposure prior to burn injury can differentially alter AM phenotype at early and later time points.
Abstract Marginal zones (MZs) are architecturally organized for clearance of and rapid response against blood-borne Ags entering the spleen. MZ macrophages (MZMs) and MZ B cells are particularly important in host defense against T-independent pathogens and may be crucial for the prevention of diseases, such as streptococcal pneumonia, that are devastating in older patients. Our objective was to determine whether there are changes in the cellular components of the MZ between old and young mice. Using immunocytochemistry and a blinded scoring system, we observed gross architectural changes in the MZs of old mice, including reduction in the abundance of MZMs surrounding the MZ sinus as well as disruptions in positioning of mucosal addressin cell adhesion molecule 1 (MAdCAM-1)+ sinus lining cells and metallophilic macrophages. Loss of frequency of MZMs was corroborated by flow cytometry. A majority of old mice also showed reduced frequency of MZ B cells, which correlated with decreased abundance of MZM in individual old mice. The spleens of old mice showed less deposition of intravenously injected dextran particles within the MZ, likely because of the decreased frequency in MZMs, because SIGN-R1 expression was not reduced on MZM from old mice. The phagocytic ability of individual MZMs was examined using Staphylococcus aureus bioparticles, and no differences in phagocytosis were found between macrophages from young or old spleens. In summary, an anatomical breakdown of the MZ occurs in advanced age, and a reduction in frequency of MZM may affect the ability of the MZM compartment to clear blood-borne Ags and mount proper T-independent immune responses.
The increasing prevalence of binge drinking and its association with trauma necessitate accurate animal models to examine the impact of intoxication on the response and outcome to injuries such as burn. While much research has focused on the effect of alcohol dose and duration on the subsequent inflammatory parameters following burn, little evidence exists on the effect of the route of alcohol administration. We examined the degree to which intoxication before burn injury causes systemic inflammation when ethanol is given by intraperitoneal (i.p.) injection or oral gavage. We found that intoxication potentiates postburn damage in the ileum, liver, and lungs of mice to an equivalent extent when either ethanol administration route is used. We also found a similar hematologic response and levels of circulating interleukin-6 (IL-6) when either ethanol paradigm achieved intoxication before burn. Furthermore, both i.p. and gavage resulted in similar blood alcohol concentrations at all time points tested. Overall, our data show an equal inflammatory response to burn injury when intoxication is achieved by either i.p. injection or oral gavage, suggesting that findings from studies using either ethanol paradigm are directly comparable.
Abstract The splenic architecture in many aged mice appears disrupted, with some similarities to the disorganization observed in mice lacking the chemokine, CXCL13. Particularly, small follicles and germinal centers are reported and the marginal zone (MZ) appears disrupted. Changes in the proportion of B cell subsets in bone marrow (BM) and secondary lymphoid tissue suggest that migration patterns may be altered. We tested the ability of young splenic B cells to migrate in an aged microenvironment. Of note, donor B cells significantly increased homing to BM in an old host. Nonetheless, similar numbers of donor follicular B cells were found in the spleens and localized to the follicles of aged vs. young recipients. However, fewer donor MZ B cells migrated into aged spleens. Unexpectedly, CXCL13 protein levels, assessed by ELISA, were greater in spleens from aged mice, but differences were apparent in the histological distribution of CXCL13 protein between young and old spleens. In old spleens, CXCL13 was unevenly distributed, concentrated in patches throughout the follicles, and reduced or absent at the follicular edge. The data suggest that CXCL13 expression may be dysregulated in aged mice. While the consequences of changes in CXCL13 distribution are under investigation, it appears not to directly affect follicular B cell migration and may have more impact on MZ B cells. NIH R01AG013874 and CCC Faculty Development Grant.
Studies in aged mice show that the architecture of B-cell areas appears disrupted and that newly made B cells fail to incorporate into the spleen. These observations may reflect altered migration of immature and mature B cells. Using adoptive transfer, we tested the effect of the aged microenvironment and the intrinsic ability of donor B cells from aged mice to migrate to spleens of intact hosts. Spleens of aged recipients were deficient in attracting young or old donor immature B cells. In contrast, immature and mature B cells maintained an intrinsic ability to migrate to young recipient spleens, except that as the aged immature B cells matured, fewer appeared to enter the recirculating pool. CXCL13 protein, which is necessary for the organization of B-cell compartments, was elevated with age and differences in CXCL13 distribution were apparent. In aged spleens, CXCL13 appeared less reticular, concentrated in patches throughout the follicles, and notably reduced in the MAdCAM-1(+) marginal reticular cells located at the follicular edge. Despite these differences, the migration of young donor follicular B cells into the spleens of old mice was not impacted; whereas, migration of young donor marginal zone B cells was reduced in aged recipients. Finally, the aged bone marrow microenvironment attracted more donor mature B cells than did the young marrow. Message for CXCL13 was not elevated in the marrow of aged mice. These results suggest that the aged splenic microenvironment affects the migration of immature B cells more than mature follicular B cells.
