Pamela L. Witte

The Role of Bone Marrow-Derived Stromal Cells in the Maintenance of Plasma Cell Longevity

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6−/− mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.

Specific recognition of apoptotic cells reveals a ubiquitous and unconventional innate immunity

The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of “self” versus “other” considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity.

Overexpression of Foxn1 attenuates age-associated thymic involution and prevents the expansion of peripheral CD4 memory T cells

The forkhead box n1 (Foxn1) transcription factor is essential for thymic organogenesis during embryonic development; however, a functional role of Foxn1 in the postnatal thymus is less well understood. We developed Foxn1 transgenic mice (Foxn1Tg), in which overexpression of Foxn1 is driven by the human keratin-14 promoter. Expression of the Foxn1 transgene increased the endogenous Foxn1 levels. In aged mice, overexpression of Foxn1 in the thymus attenuated the decline in thymocyte numbers, prevented the decline in frequency of early thymic progenitors, and generated a higher number of signal joint TCR excised circle. Histologic studies revealed that structural alterations associated with thymic involution were diminished in aged Foxn1 Tg. Total numbers of EpCAM+ MHC II+ and MHC II(hi) thymic epithelial cells were higher in young and old Foxn1Tg and more EpCAM+ MHC II(hi) TEC expressed Ki-67 in aged Foxn1Tg compared with WT. Furthermore, Foxn1Tg displayed a significant reduction in the expansion of splenic CD4+ memory compartments and attenuated the decline in CD4+ and CD8+ naive compartments. Our data indicate that manipulation of Foxn1 expression in the thymus ameliorates thymopoiesis in aged mice and offer a strategy to combat the age-associated decline in naive T-cell production and CD4 naive/memory ratios in the elderly.

Alterations in Marginal Zone Macrophages and Marginal Zone B Cells in Old Mice

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.

CD1d-Restricted NKT Cells Contribute to the Age-Associated Decline of T Cell Immunity

NKT cells are known to regulate effector T cell immunity during tolerance, autoimmunity, and antitumor immunity. Whether age-related changes in NKT cell number or function occur remains unclear. Here, we investigated whether young vs aged (3 vs 22 mo old) mice had different numbers of CD1d-restricted NKT cells and whether activation of NKT cells by CD1d in vivo contributed to age-related suppression of T cell immunity. Flow cytometric analyses of spleen and LN cells revealed a 2- to 3-fold increase in the number of CD1d tetramer-positive NKT cells in aged mice. To determine whether NKT cells from aged mice differentially regulated T cell immunity, we first examined whether depletion of NK/NKT cells affected the proliferative capacity of splenic T cells. Compared with those from young mice, intact T cell preparations from aged mice had impaired proliferative responses whereas NK/NKT-depleted preparations did not. To examine the specific contribution of NKT cells to age-related T cell dysfunction, Ag-specific delayed-type hypersensitivity and T cell proliferation were examined in young vs aged mice given anti-CD1d mAb systemically. Compared with young mice, aged mice given control IgG exhibited impaired Ag-specific delayed-type hypersensitivity and T cell proliferation, which could be significantly prevented by systemic anti-CD1d mAb treatment. The age-related impairments in T cell immunity correlated with an increase in the production of the immunosuppressive cytokine IL-10 by splenocytes that was likewise prevented by anti-CD1d mAb treatment. Together, our results suggest that CD1d activation of NKT cells contributes to suppression of effector T cell immunity in aged mice.

Aging enhances lymphocyte cytokine defects after injury

Mortality and sepsis after a traumatic injury is greater in the elderly than in young individuals. The altered lymphocyte response observed to occur in healthy aged individuals is proposed to be a contributing factor to increased mortality. The immune response associated with the increased mortality was explored using a murine scald injury model. In the absence of injury, aged mice had depressed delayed‐type hypersensitivity (DTH) and splenocyte proliferative responses relative to young mice. There was also an increase with age in the production of the TH2 cytokines interleukin (IL)‐4 and IL‐10 by splenocytes. There was no change in the TH1 cytokines IFNγ or IL‐12 with age. However, IL‐2 production was significantly lower. Following injury, there was a further decrease in the DTH response of aged injured mice, compared with aged sham mice. In addition, there was a decrease in all of the cytokines examined, regardless of age. In contrast, IFNγ and IL‐2 were significantly lower in the aged injured animals compared with the young injured animals. These results suggest that the lack of an adequate amount of TH1 cytokines shortly after injury in the aged mice may parallel the increased incidence of sepsis and death that occurs in aged burn patients.

Estrogen replacement, aging, and cell-mediated immunity after injury

We recently demonstrated that aged mice are less likely to survive following traumatic injury and are more immunosuppressed than young mice who sustain comparable injuries. Immunosuppression in severely injured patients and in rodent models of burn injury is associated with a marked elevation in proinflammatory cytokines, including interleukin‐6 (IL‐6). We reported that after sustaining a moderate‐size scald injury, aged mice have higher circulating levels of IL‐6 than young, injured mice. As proestrus levels of estrogen have been reported to boost immune responses and attenuate IL‐6 production, in the present study, we went on to determine if estrogen replacement in aged female mice restored cellular immunity and proinflammatory cytokine production. After injury, in placebo‐treated, aged animals, there was a >75% suppression in the delayed‐type hypersensitivity response relative to placebo‐treated, sham‐injured, aged mice (P<0.05). In contrast, estrogen supplementation before injury yielded a partial recovery in this response, such that the mice were suppressed by only 40% relative to sham‐injured, aged mice (P<0.05). There was a fourfold increase in the circulating level of IL‐6 in burn‐injured, aged mice who received placebo hormone replacement relative to sham‐injured mice given placebo (P<0.05). This level of cytokine was lowered by nearly 50% in aged, estrogen‐treated mice. Most remarkably, estrogen replacement improved survival from 42% (in the absence of estrogen) to 70% in aged, burn‐injured mice. Further investigation will be needed to determine if age‐ and gender‐specific therapies are needed for the treatment of all trauma patients.

Migration of immature and mature B cells in the aged microenvironment.

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.

Expression of CD28 by Bone Marrow Stromal Cells and Its Involvement in B Lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2–6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15–20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28−/− mice were similar to those in wild-type mice; however, older CD28−/− mice (15–19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.

Role of stromal cells and their products in protecting young and aged B-lineage precursors from dexamethasone-induced apoptosis

Bone marrow stromal cells are potent providers of stimuli that induce proliferation of B-cell precursors. We proposed that stromal cells play a role in protecting B-lineage cells from corticosteroid-induced apoptosis. We found that stromal cells protected B-cell precursors from dexamethasone-induced apoptosis, but this did not strictly correlate with interleukin-7 (IL-7) production. To determine if stromal-derived factors were involved in protection of B-cell precursors from apoptosis, we examined the activity of three lymphopoietic growth factors: IL-7, stem cell factor (SCF), and insulin-like growth factor-1 (IGF-1). Either IL-7 or IGF-1 alone protected B-cell precursors from dexamethasone-induced apoptosis. The combined activities of IGF-1 and IL-7 were additive rather than synergistic. SCF did not protect B-cell precursors from apoptosis. Aging altered the ability of B-cell precursors to respond to protective stimuli induced by IL-7 and IGF-1. Precursors from aged animals were deficient in ability to modulate expression of apoptosis regulatory genes Bax, Bcl-2, and Bcl-x in comparison to B-cell precursors from young animals. Taken together, these results suggest that stromal cells can protect B-lineage precursors from a corticosteroid-induced apoptotic signal, protection is mediated by stromal-derived cytokines, and aging decreases the ability of B-cell precursors to respond efficiently to protective stimuli.