Vascular cell adhesion molecule-1-targeted detection of endothelial activation in human microvasculature.

The hallmark of endothelial activation, an early and critical step in many alloimmune and inflammatory responses, is the transcriptional induction and expression of endothelial adhesion molecules (eg, vascular cell adhesion molecule-1 [VCAM-1]). We assessed the feasibility of VCAM-1-targeted in vivo detection of endothelial activation using I-125-labeled-F(ab')2 fragments of E1/6, a monoclonal antibody against human but not murine VCAM-1. The Kd and Bmax, determined by saturation binding in tumor necrosis factor (TNF)-activated human endothelial cells (ECs), were 3.2 +/- 0.6 nmol/L and 5600 +/- 300 binding sites per EC, respectively. Biodistribution and in vivo binding characteristics of I-125-E1/6 F(ab')2 were assessed in a novel chimeric human/mouse model, in which human skin (as a source of human microvasculature) is grafted onto SCID/beige mice. I-125-E1/6 F(ab')2 localized to TNF-activated human skin grafts as detected by autoradiography and gamma well-counting. Relative uptakes (uptake in human skin graft/uptake in the surrounding mouse skin) were, respectively, 2.6 +/- 0.8 (n = 14) and 1.6 +/- 0.3 (n = 12) for E1/6 and MOPC-21, an isotype-matched control antibody (P < .01). The preferential uptake in human skin graft was not due to differences in tissue vascularity assessed by Tc-99m-labeled murine red blood cells. In conclusion, the chimeric human/mouse model is a novel experimental tool for in vivo evaluation of human endothelial cell-specific radiopharmaceuticals. Although I-125-E1/6 F(ab')2 localized to human skin grafts, the limited number of VCAM-1 molecules/endothelial cell adversely affects its suitability as a target for in vivo imaging of endothelial activation.

Human endothelial cell presentation of antigen and the homing of memory/effector T cells to skin.

Dermal microvascular endothelial cells (ECs) form a continuous lining that normally bars blood-borne T lymphocytes from entering the skin, but as part of the response to foreign antigen, dermal ECs undergo alterations in their surface proteins so as to provide signals to circulating T cells that lead to their activation and recruitment. Several observations suggest that human dermal microvascular ECs may help initiate cutaneous immune reactions by presentation of cognate antigens to circulating T memory cells: (1) antigen-specific inflammatory responses in the skin, as in other organs, involve accumulation of memory and effector T cell populations that are enriched in cells specific for the eliciting antigen; (2) recall responses to intradermal protein antigens in the skin start very rapidly within two hours of challenge; (3) dermal microvascular ECs in humans and other large mammals basally display high levels of class I and class II MHC molecules, the only known purpose of which is to present antigenic peptides to lymphocytes; (4) the lumen of dermal capillaries are narrower than the diameter of circulating T cells, ensuring surface contact; and (5) cultured human ECs effectively present antigens to resting memory T cells isolated from the circulation. Upon contact with activated T cells or their secreted products (cytokines), dermal ECs themselves become activated, increasing their capacity to recruit memory and effector T cell populations in an antigen-independent manner. Specifically, activated ECs express inducible leukocyte adhesion molecules such as E-selectin, ICAM-1, and VCAM-1; and several lines of evidence, including neutralizing antibody experiments and gene knockouts, have supported a role of these molecules in T cell recruitment. Dermal ECs have unique expression patterns of adhesion molecules that can determine the subsets of memory T cells that are recruited into the skin. For example, slow internalization of E-selectin allows more persistent expression of this protein on the surface of dermal ECs, favoring interactions with CLA-1+ T cells. VCAM-1 expression, normally confined to venular EC may extend to capillaries within the dermal papillae and contribute to epidermal inflammation, recruiting alpha4beta7 integrin-expressing T cells that also express the cadherin-binding integrin alphaEbeta7. New models involving transplantation of normal and genetically modified human dermal ECs into immunodeficient mice may be used to further explore these properties.

Human T cells infiltrate and injure pig coronary artery grafts with activated but not quiescent endothelium in immunodeficient mouse hosts.

BACKGROUND: We have previously demonstrated that human artery grafts transplanted to immunodeficient mice are infiltrated and injured by unsensitized allogeneic human T cells. We extended our investigations to human anti-porcine xenoresponses in this model. METHODS: Pig coronary artery segments were interposed into the infrarenal aorta of severe combined immunodeficiency/beige mice. After 7 days, certain recipients were reconstituted with human leukocytes and/or treated with proinflammatory cytokines. The grafts were harvested after 1-70 days and examined by histology, immunohistochemistry, and morphometry. RESULTS: Pig artery grafts from untreated mice had no evidence of injury, leukocytic infiltrate, or endothelial cell activation up to 70 days postoperatively, despite deposition of murine complement. Host reconstitution with human peripheral blood mononuclear cells resulted in a discrete population of circulating T cells that did not infiltrate or injure the grafts up to 28 days after adoptive transfer. Administration of porcine interferon-gamma for up to 28 days sustained the expression of graft vascular cell adhesion molecule-1 and major histocompatibility complex antigens, but did not initiate recruitment of human leukocytes. In contrast, treatment with human tumor necrosis factor for 7 days induced the de novo expression of porcine E-selectin by graft endothelial cells and elicited human T cell infiltration and human peripheral blood mononuclear cell-dependent vascular injury. CONCLUSIONS: The human peripheral blood mononuclear cell-severe combined immunodeficiency/beige mouse model identifies a significant difference between human T cell allogeneic and xenogeneic responses in vivo. Xenografts with quiescent endothelium are not infiltrated or injured by T cells under the same conditions in which allografts are rejected. Activation of pig coronary artery endothelial cells by human tumor necrosis factor, but not porcine interferon-gamma, elicits cellular xenoresponses.

Inhibition of melanoma tumor growth in vivo by survivin targeting.

A role of apoptosis (programmed cell death) in tumor formation and growth was investigated by targeting the apoptosis inhibitor survivin in vivo. Expression of a phosphorylation-defective survivin mutant (Thr(34)-->Ala) triggered apoptosis in several human melanoma cell lines and enhanced cell death induced by the chemotherapeutic drug cisplatin in vitro. Conditional expression of survivin Thr(34)-->Ala in YUSAC2 melanoma cells prevented tumor formation upon s.c. injection into CB.17 severe combined immunodeficient-beige mice. When induced in established melanoma tumors, survivin Thr(34)-->Ala inhibited tumor growth by 60-70% and caused increased apoptosis and reduced proliferation of melanoma cells in vivo. Manipulation of the antiapoptotic pathway maintained by survivin may be beneficial for cancer therapy.

In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.

We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the gel 31 days after implantation. Retroviral-mediated overexpression of a caspase-resistant Bcl-2 protein delays HUVEC apoptosis in vitro for over 7 days. Bcl-2-transduced HUVEC produce an increased density of HUVEC-lined perfused microvessels in vivo compared with untransduced or control-transduced HUVEC. Remarkably, Bcl-2- but not control-transduced HUVEC recruit an ingrowth of perivascular smooth-muscle alpha-actin-expressing mouse cells at 31 days, which organize by 60 days into HUVEC-lined multilayered structures resembling true microvessels. This system provides an in vivo model for dissecting mechanisms of microvascular remodeling by using genetically modified endothelium. Incorporation of such human endothelial-lined microvessels into engineered synthetic skin may improve graft viability, especially in recipients with impaired angiogenesis.

Human TNF can induce nonspecific inflammatory and human immune-mediated microvascular injury of pig skin xenografts in immunodeficient mouse hosts.

TNF activates endothelial cells to express cell surface molecules that are necessary to recruit a local infiltrate of leukocytes. Because the actions of this proinflammatory cytokine are not species restricted, we investigated whether human TNF can up-regulate porcine endothelial adhesion molecules to elicit human T cell infiltration and damage of pig skin xenografts in a chimeric immunodeficient mouse model. We have previously demonstrated the vigorous rejection of human skin allografts and the absence of injury to porcine skin xenografts in human PBMC-SCID/beige mice. Intradermal administration of human TNF at high doses (600 or 2000 ng) caused nonspecific inflammatory damage of pig skin grafts, whereas low concentrations of TNF (60 or 200 ng) resulted in human PBMC-dependent injury of porcine endothelial cells. There was a strong correlation among pig skin xenograft damage, human T cell infiltration, and the TNF-induced up-regulation of swine MHC class I and class II molecules, VCAM-1, and, in particular, the de novo expression of porcine E-selectin. The microvascular damage and leukocytic infiltration elicited by TNF were enhanced by porcine IFN-gamma, suggesting that xenografts may be less prone to cytokine-mediated injury due to the species-restricted effects of recipient IFN-gamma. Our results indicate that maintenance of a quiescent endothelium, which does not express E-selectin or other activation-dependent adhesion molecules, is important in preventing human anti-porcine T cell xenoresponses in vivo and that TNF signaling molecules and TNF-responsive gene products are appropriate therapeutic targets to protect against human T cell-mediated rejection of pig xenografts.

Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses.

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-kappaB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Control of apoptosis during angiogenesis by survivin expression in endothelial cells.

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.

Interferon-gamma elicits arteriosclerosis in the absence of leukocytes.

Atherosclerosis and post-transplant graft arteriosclerosis are both characterized by expansion of the arterial intima as a result of the infiltration of mononuclear leukocytes, the proliferation of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular matrix. They are also associated with the presence of the immunomodulatory cytokine interferon-gamma (IFN-gamma). Moreover, in mouse models of atheroma formation or allogeneic transplantation, the serological neutralization or genetic absence of IFN-gamma markedly reduces the extent of intimal expansion. However, other studies have found that exogenous IFN-gamma inhibits cultured VSMC proliferation and matrix synthesis, and reduces intimal expansion in response to mechanical injury. This discrepancy is generally explained by the idea that IFN-gamma either directly activates macrophages, or, by increasing antigen presentation, indirectly activates T cells within the lesions of atherosclerosis and graft arteriosclerosis. These activated leukocytes are thought to express the VSMC-activating cytokines and cell-surface molecules that cause the observed arteriosclerotic responses. Here we have inserted pig and human arteries into the aorta of immunodeficient mice, and we show that IFN-gamma can induce arteriosclerotic changes in the absence of detectable immunocytes by acting on VSMCs to potentiate growth-factor-induced mitogenesis.

Integrins alpha4beta7 and alphaEbeta7 are expressed on epidermotropic T cells in cutaneous T cell lymphoma and spongiotic dermatitis.

Integrin alpha4beta7 has been associated with tissue-specific homing of malignant and inflammatory lymphocytes to gastrointestinal mucosa, whereas integrin alphaEbeta7 has been associated with intraepithelial lymphocytes in both the gut and the skin. This prompted us to examine the expression of alpha4beta7 on skin-infiltrating lymphocytes in 12 cases of patch/plaque stage cutaneous T cell lymphoma (CTCL) and in 4 cases of spongiotic dermatitis, which also display intraepidermal T cell accumulation. alpha4beta7 was found to be expressed on 64.8+/-7.4% of intraepidermal and 39.1+/-5.0% of intradermal T lymphocytes in CTCL. There was a significant positive correlation (r=0.58) between the degree of epidermotropism and the percentage of intraepidermal T cells expressing alpha4beta7. Similar findings were observed in spongiotic dermatitis, indicating that this result is not unique to malignant T cells. We evaluated staining of T cells in the same specimens for presence of alphaEbeta7 and observed a strong correlation between the expression of both beta7 integrins in each specimen. Staining with antibodies directed against the known ligands of alpha4beta7 was also performed on skin biopsies from CTCL patients. There was significantly increased dermal microvascular endothelial expression of vascular cell adhesion molecule-1 in lesional compared with nonlesional skin, and in nonlesional skin compared with skin of normal control subjects. Dermal and epidermal expression of the CS-1 domain of fibronectin was present but not increased in lesional biopsies compared with nonlesional or normal controls, whereas expression of mucosal addressin cell adhesion molecule-1 was not detectable in any skin biopsy specimens. In summary, alpha4beta7, like alphaEbeta7, is expressed at high levels on epidermotropic T cells and may interact with endothelial cell vascular cell adhesion molecule-1 as part of stepwise recruitment of lymphocytes from the blood to the epidermis.

Human allogeneic vascular rejection after arterial transplantation and peripheral lymphoid reconstitution in severe combined immunodeficient mice.

BACKGROUND: Interspecies differences create important shortcomings in existing animal models used to describe in vivo events responsible for allograft rejection. Alloimmune destruction of human dermal microvessels, histologically consistent with rejection, has been demonstrated in human skin-grafted severe combined immunodeficient (SCID) mice receiving allogeneic human peripheral blood mononuclear cells (PBMC). We have now documented human alloimmune injury in a vascularized, SCID-human arterial transplantation model. METHODS: Fresh human artery was used to replace the CB.17 SCID/beige mouse infrarenal aorta. Seven days later, 3x10(8) human PBMC were administered intraperitoneally, and lymphocyte engraftment was considered successful when circulating human CD3+ cells were later identified in peripheral blood. RESULTS: Forty-six of 49 (94%) mice undergoing transplantation survived, including 14 controls with arterial grafts receiving no PBMC. Twenty-eight of 32 mice demonstrated circulating human CD3+ cells, 14 days after PBMC administration. Animals were killed at 14, 21, or 28 days after receiving allogeneic PBMC, and arteries were recovered for histology and immunohistology. All 14 control mice had patent transplanted grafts with normal vascular histology and no lymphoid infiltration. Damage to transplanted arteries among lymphocyte-engrafted mice was apparent by 14 and 21 days in some animals, whereas 16 of 22 exhibited moderate to severe intimal, medial, and/or adventitial lymphocytic infiltration with intimal expansion by day 28. The infiltrate consisted of HLA-A, -B, -C+, and -DR+, human CD3+ cells, approximately equally distributed as CD4+ and CD8+ subsets. Some infiltrating lymphocytes were cytolytic cells as demonstrated by perforin staining. The endothelium of transplanted human arteries exhibited endothelialitis, and the endothelial cells stained intensely with anti-HLA-A, -B, -C and anti-HLA-DR antibodies. The expanded intima was predominantly smooth muscle cells, staining positively for smooth muscle alpha-actin, HLA-A, -B, -C and HLA-DR. Medial necrosis was not observed. CONCLUSION: The results provide evidence of alloimmune-mediated vascular rejection in this human arterial transplantation model.

Biological action of leptin as an angiogenic factor.

Leptin is a hormone that regulates food intake, and its receptor (OB-Rb) is expressed primarily in the hypothalamus. Here, it is shown that OB-Rb is also expressed in human vasculature and in primary cultures of human endothelial cells. In vitro and in vivo assays revealed that leptin has angiogenic activity. In vivo, leptin induced neovascularization in corneas from normal rats but not in corneas from fa/fa Zucker rats, which lack functional leptin receptors. These observations indicate that the vascular endothelium is a target for leptin and suggest a physiological mechanism whereby leptin-induced angiogenesis may facilitate increased energy expenditure.

Dermal microvascular injury in the human peripheral blood lymphocyte reconstituted-severe combined immunodeficient (HuPBL-SCID) mouse/skin allograft model is T cell mediated and inhibited by a combination of cyclosporine and rapamycin.

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.

Mycosis fungoides palmaris et plantaris or acral pagetoid reticulosis?

There has been ongoing debate about the nature of Woringer-Kolopp disease (unilesional pagetoid reticulosis). Despite the histologic resemblance to mycosis fungoides, these lesions are typically solitary and indolent. Recently, cutaneous plaques of epidermotropic lymphocytes restricted to acral sites resembling Woringer-Kolopp disease were reported to show T-cell clonality, leading to the designation mycosis fungoides palmaris et plantaris. We describe a similar case of recurrent plaques on palms and soles of a 45-year-old man that persisted for >14 years without other cutaneous or systemic disease. Histologically, the lesions were comprised of epidermotropic atypical lymphocytes with sparse dermal infiltrates. Immunohistochemically, the majority of intraepidermal lymphocytes labeled as CD8-positive suppressor/cytotoxic T cells and expressed alphaE beta7 (CD103), an integrin associated with epitheliotropism. Polymerase chain reaction studies revealed similar clonal gene rearrangements of T-cell receptors beta and gamma in tissue from both palm and sole. In view of these findings, the diagnosis of mycosis fungoides palmaris et plantaris may be appropriate. To date, however, the lesions have remained localized and continue to resolve spontaneously. As such, the behavior is similar to what has been described as pagetoid reticulosis. Long-term follow-up will be necessary to determine the biologic potential of this disease.

Blockade of CD2-LFA-3 interactions protects human skin allografts in immunodeficient mouse/human chimeras.

A human skin allograft injury model in immunodeficient mice, engrafted with human peripheral blood mononuclear cells from a different donor, has been used to test whether reagents that block human T cell CD2 interactions with its principal ligand, LFA-3 (CD58), can inhibit immune reactions in vivo. In this model, human skin grafts show a reproducible pattern of progressive human T-cell infiltration and human graft microvascular injury that resembles human first-set skin graft rejection. Murine Mab to human LFA-3 or human LFA-3-IgG1 fusion protein, but not isotype-matched control antibodies, each markedly protected skin grafts from leukocyte infiltration and injury. These data provide the first evidence that LFA-3 functions in vivo and establish the ability of this new model to test human-specific immune modulators.

Topographic mapping of the cutaneous microcirculation using two outputs of laser-Doppler flowmetry: flux and the concentration of moving blood cells.

Using a "needle" probe in a template probe holder, we measured the flux and the concentration of moving red blood cell (CMBC) outputs from a Perimed (PF2B) laser-Doppler instrument at 1-mm2 contiguous sites in a 8 x 8-mm area on the flexor forearm of three subjects. Using the means of the flux and CMBC recorded at each spot, a topographic contour map was constructed for each of these parameters. Viewing the two maps together, sites with four different combinations of flux and CMBC could be identified. Trephine biopsies (2 mm) of three representative sites in each subject were performed and the upper plexus was reconstructed in 3 dimensions from serial sections. High flux/high-to-medium CMBC sites were found over the spot where the ascending arterioles entered the upper plexus. Medium flux/medium-to-low CMBC sites and low flux/medium CMBC sites were found in the peripheral part of the vascular unit that was fed by the ascending arteriole. The low flux/low CMBC sites were relatively avascular zones. Video imaging of the upper plexus in the forearm showed the same overall vascular pattern as the contour maps. The highest flux and CMBC signals were recorded when horizontally oriented vessels were present in the upper third of the plexus (400-650 microns below the stratum corneum). Topographic mapping will allow one to selectively identify different microvascular areas in the skin for physiological studies.