Douglas A. Carlow

Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model.

Objective: To establish the generality of cerebrovascular pathology frequently observed with Alzheimer disease, we have assessed blood‐brain barrier (BBB) integrity using the Alzheimer disease model Tg2576 mice in which cognitive deficits and neuritic plaque formation develop around 10–12 months of age.

PSGL‐1 function in immunity and steady state homeostasis

Summary:  The substantial importance of P‐selectin glycoprotein ligand 1 (PSGL‐1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL‐1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin‐PSGL‐1 interaction requires branched O‐glycan extensions on specific PSGL‐1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O‐glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL‐1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O‐glycans on PSGL‐1 and provide the molecular interactions responsible for the velcro‐like catch bonds that support leukocyte rolling. Such binding of PSGL‐1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL‐1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T‐cell homing to secondary lymphoid organs. PSGL‐1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O‐glycan modifications of PSGL‐1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL‐1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T‐cell traffic. The multi‐level influence of PSGL‐1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O‐glycans. However, central as specific O‐glycosylation is to PSGL‐1 function, in vivo regulation of PSGL‐1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL‐1 glycosylation and to update understanding of PSGL‐1 functional scope.

Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs

P-selectin glycoprotein ligand 1 (PSGL-1) is central to the trafficking of immune effector cells to areas of inflammation through direct interactions with P-selectin, E-selectin and L-selectin. Here we show that PSGL-1 was also required for efficient homing of resting T cells to secondary lymphoid organs but functioned independently of selectin binding. PSGL-1 mediated an enhanced chemotactic T cell response to the secondary lymphoid organ chemokines CCL21 and CCL19 but not to CXCL12 or to inflammatory chemokines. Our data show involvement of PSGL-1 in facilitating the entry of T cells into secondary lymphoid organs, thereby demonstrating the bifunctional nature of this molecule.

IL-2, -4, and -15 differentially regulate O-glycan branching and P-selectin ligand formation in activated CD8 T cells

The glycosyltransferase core 2 β1–6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8+ T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8+ cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.

Absence of CD43 fails to alter T cell development and responsiveness

Genetic elimination of CD43 has been associated with increased T cell adhesiveness and T cell hyperresponsiveness to mitogens and alloantigens. Therefore, we investigated whether T cell development was perturbed in CD43-deficient mice by breeding CD43null mice with male Ag (Hy)-specific TCR-transgenic mice. Neither positive nor negative thymic selection of male Ag-specific T cells were affected by CD43 status. Furthermore, we did not observe a substantial or consistent hyperresponsive pattern in HY-CD43null lymph node cells compared with littermate HY-CD43+/− lymph node cells upon analysis of in vitro T cell stimulation with male Ag or mitogen. These observations challenged original conclusions associating absence of CD43 with T cell hyperresponsiveness and led us to re-examine this association. Reported phenotypes of CD43null mice have been based on mice with a mixed 129×C57BL/6 genetic background. To exclude a possible influence of genetic background differences among individual mice we analyzed CD43null littermates that had been back-bred onto the C57BL/6 background for seven to eight generations. We found that CD43+ and CD43null littermates with the C57BL/6 background exhibited no differences in response to mitogen or alloantigen, thereby establishing that T cell hyperresponsiveness is not a general correlate of CD43 absence.

Lymphocytes in the Peritoneum Home to the Omentum and Are Activated by Resident Dendritic Cells

The omentum is of interest in the context of obesity-related metabolic disease where adipose tissue exhibits inflammatory changes; however, the immunology of the omentum is underexplored. The greater omentum is draped from the stomach and consists predominantly of adipose tissue studded with lymphoreticular aggregations (milky spots) that distinguish it from other visceral adipose tissues. Milky spots are thought to contain and conduct leukocytes in transit from the blood to the peritoneal cavity, particularly during peritonitis. We show here that both B and T lymphocytes counterflow from the peritoneal cavity to the omentum in mice. Residence in the omentum was brief with a t1/2 residence time of 6 h. Omentum access was pertussis toxin-sensitive, dependent on activation of the Rap1 GTPase, and on the integrin LFA-1. B cells and CD44high T cells accessed the omentum most efficiently, but homing of resting CD44low T cells was also observed. Omental tissue from normal healthy mice was found to contain CD8−CD11bhighMHC class IIhighCD11chigh dendritic cells that promoted the rapid activation of T cells entering the omentum and cross-presented soluble OVA or OVA acquired from either OVA-expressing Escherichia coli or OVA-pulsed spleen cells. We conclude that the omentum incorporates two key features of immunological sentinel function, actively supported lymphocyte traffic and dendritic cells, that reinforce a conceptual framework for function in stimulating adaptive immunity. These results extend basic understanding of omental and peritoneal cavity immunology and of how proinflammatory events occurring within the peritoneal cavity might affect adipocyte and hepatocyte metabolism.

CD43 Deficiency Has No Impact in Competitive In Vivo Assays of Neutrophil or Activated T Cell Recruitment Efficiency

Using noncompetitive methodologies comparing CD43+/+ and CD43−/− mice, it has been reported that CD43−/− leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43−/− T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43+/+ vs CD43−/− activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43+/+ and congenic CD43−/− mice in either B6 or P-selectin−/− recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

Modulation of O-glycans and N-glycans on murine CD8 T cells fails to alter annexin V ligand induction by galectin 1.

Thymic negative selection and contraction of responding T cell oligoclones after infection represent important cell ablation processes required for maintaining T cell homeostasis. It has been proposed that galectin 1 contributes to these processes through interaction with lactosyl sequences principally on cell surface glycoproteins bearing core 2 (C2GnT1)-branched O-glycans. According to this model, specific T cell surface proteins cross-linked by galectin 1 induce signaling, ligand redistribution, and apoptosis in both immature thymocytes and activated T cells. The influence of lactosyl residues contained in branched O-glycans or complex N-glycans on galectin 1 binding and induction of annexin V ligand in murine CD8 T cells was assessed. Neither galectin binding nor galectin-induced expression of annexin V ligand was perturbed under conditions in which: 1) C2GnT1 activity was differentially induced by CD8 T cell activation/culture with IL-2 vs IL-4; 2) activated CD8+ T cells lacked C2GnT1 expression; or 3) complex N-glycan formation was blocked by swainsonine. The maintenance of galectin 1 binding and induced annexin V expression under conditions that alter lactosamine abundance on O- or complex N-glycans suggest that galectin 1-mediated apoptosis is neither a simple function of fluctuating C2GnT1 activity nor a general C2GnT1-dependent mechanism underlying contraction of CD8 T cells subsequent to activation.

Inducing P-Selectin Ligand Formation in CD8 T Cells: IL-2 and IL-12 Are Active In Vitro but Not Required In Vivo

In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2null CD8 T cells responding in doubly deficient IL-2nullIL-12null or IL-2nullIL-15null male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.

PSGL-1 Regulates the Migration and Proliferation of CD8+ T Cells under Homeostatic Conditions

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1null mice. Naive CD4+ and CD8+ T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1null T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1+/+ T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1null CD8+ T cell proliferation was observed under steady-state conditions and PSGL-1null CD8+ T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1null mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1null mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1null mice.