Richard D. Granstein

Dendritic cells as initiators of tumor immune responses: a possible strategy for tumor immunotherapy?

Abstract Since all T-cell-mediated immune responses require antigen presentation, antigen-presenting cells (APCs) may be of central importance for the generation and regulation of tumor immunity. This article describes approaches used to induce tumor immunity via modulation of the presentation of tumor antigen, either by tumor-antigen-exposed dendritic cells or by tumor cells engineered to act as APCs.

Gamma-interferon inhibits collagen synthesis in vivo in the mouse.

Subcutaneous implantation of osmotic pumps into CAF1 mice resulted in the formation of thick fibrous capsules around the pumps. When pumps were loaded with recombinant murine gamma-interferon (rMuIFN-gamma) to deliver 2 X 10(3) U/h for 14 d, there was a marked decrease in thickness and collagen content of the capsules from rMuIFN-gamma-treated animals compared with capsules from animals receiving diluent alone. The collagen content of the capsules was estimated by hydroxyproline analysis of the tissue and by quantitative electron microscopy of collagen bundles. Heat-inactivated rMuIFN-gamma failed to reduce the fibrotic response in this assay. These results provide compelling evidence that gamma-interferon can down-regulate collagen synthesis in vivo and suggest the possibility that this lymphokine may be useful in the treatment of disease states characterized by excessive fibrosis.

Drug- and heavy metal-induced hyperpigmentation

Several categories of chemical and pharmacologic agents can cause alterations in cutaneous pigmentation, although the mechanisms differ and in several instances may be unknown. Fixed drug eruptions appear to have alteration of the basement membrane zone with incontinence of epidermal pigment as the mechanism of hyperpigmentation. Heavy metals produce increased pigmentation in part from deposition of metal particles and in part from an increase in epidermal melanin production. The antimalarials may bind to melanin. The phenothiazines and minocycline produce pigmentation from deposition of the drug. The mechanism, site, and nature of the pigment occurring with antineoplastic agents is not well understood, but the location is most likely predominantly epidermal. Clofazimine (Lamprene) alteration in pigmentation appears to result from deposition of the drug in subcutaneous fat.

Cathelicidin Antimicrobial Peptides Block Dendritic Cell TLR4 Activation and Allergic Contact Sensitization

Cathelicidins are antimicrobial peptides of the innate immune system that establish an antimicrobial barrier at epithelial interfaces and have been proposed to have a proinflammatory function. We studied the role of cathelicidin in allergic contact dermatitis, a model requiring dendritic cells of the innate immune response and T cells of the adaptive immune response. Deletion of the murine cathelicidin gene Cnlp enhanced an allergic contact response, whereas local administration of cathelicidin before sensitization inhibited the allergic response. Cathelicidins inhibited TLR4 but not TLR2 mediated induction of dendritic cell maturation and cytokine release, and this inhibition was associated with an alteration of cell membrane function and structure. Further analysis in vivo connected these observations because inhibition of sensitization by exogenous cathelicidin was dependent on the presence of functional TLR4. These observations provide evidence that cathelicidin antimicrobial peptides mediate an anti-inflammatory response in part by their activity at the membrane.

Regulation of cytokine expression in macrophages and the Langerhans cell-like line XS52 by calcitonin gene-related peptide.

Calcitonin gene‐related peptide (CGRP) inhibits antigen presentation by Langerhans cells (LC) and macrophages, and LC are anatomically associated with CGRP‐containing epidermal nerves. To determine whether CGRP may produce some of its functional effects through regulation of cytokine expression, we utilized enzyme‐linked immunosorbent assay (ELISA) of conditioned supernatants to examine production of interleukin (IL)‐10 and IL‐1β protein in the LC‐like cell line XS52 as well as the reverse transcriptase‐polymerase chain reaction (RT‐PCR) to examine levels of mRNA for IL‐10, IL‐1β, and the 40‐kDa subunit (p40) of IL‐12. CGRP augmented the lipopotysaccharide (LPS) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) ‐induced release of IL‐10 protein and the induced expression of IL‐10 mRNA in these cells. However, it suppressed the induction of release of IL‐1β protein and the induction of mRNA for IL‐12 p40 and IL‐1β by LPS and GM‐CSF. Regulation of cytokine expression in peritoneal macrophages was also examined. By ELISA, the LPS‐induced expression of IL‐10 was augmented by CGRP, whereas the induction of IL‐1β was suppressed. Northern analysis demonstrated augmentation of LPS‐induced IL‐10 mRNA levels and inhibition of LPS‐induced IL‐1β mRNA by CGRP. CGRP inhibited the LPS‐induced induction of IL‐12 mRNA as assessed by RT‐PCR. Up‐regulation of B7‐2 expression by LPS and GM‐CSF was suppressed by CGRP in both XS52 cells and macrophages, as previously reported. This suppression, however, could be abrogated by co‐culture with neutralizing antibodies to IL‐10. Furthermore, the presence of neutralizing antibodies to IL‐10 during exposure of epidermal cells (EC) to CGRP prevented the CGRP‐mediated suppression of EC presentation of tumor‐associated antigens (from the S1509a spindle cell carcinoma) for elicitation of delayed‐type hypersensitivity in S1509a‐immune mice. These data suggest that suppression of antigen‐presenting function by CGRP is mediated, at least in part, by changes in cytokine expression that favor less robust antigen presentation for cell‐mediated immunity. J. Leukoc. Biol. 61: 216–223; 1997.

Catecholamines Inhibit the Antigen-Presenting Capability of Epidermal Langerhans Cells

The sympathetic nervous system modulates immune function at a number of levels. Within the epidermis, APCs (Langerhans cells (LC)) are frequently anatomically associated with peripheral nerves. Furthermore, some neuropeptides have been shown to regulate LC Ag-presenting function. We explored the expression of adrenergic receptors (AR) in murine LC and assessed their functional role on Ag presentation and modulation of cutaneous immune responses. Both purified LC and the LC-like cell lines XS52-4D and XS106 expressed mRNA for the ARs α1A and β2. XS106 cells and purified LC also expressed β1-AR mRNA. Treatment of murine epidermal cell preparations with epinephrine (EPI) or norepinephrine inhibited Ag presentation in vitro. Furthermore, pretreatment of epidermal cells with EPI or norepinephrine in vitro suppressed the ability of these cells to present Ag for elicitation of delayed-type hypersensitivity in previously immunized mice. This effect was blocked by use of the β2-adrenergic antagonist ICI 118,551 but not by the α-antagonist phentolamine. Local intradermal injection of EPI inhibited the induction of contact hypersensitivity to epicutaneously administered haptens. Surprisingly, injection of EPI at a distant site also suppressed induction of contact hypersensitivity. Thus, catecholamines may have both local and systemic effects. We conclude that specific ARs are expressed on LC and that signaling through these receptors can decrease epidermal immune reactions.

In vivo inflammatory activity of epidermal cell-derived thymocyte activating factor and recombinant interleukin 1 in the mouse.

Epidermal cell-derived thymocyte activating factor (ETAF), a cytokine produced by keratinocytes, has previously been shown to be biochemically and functionally very similar, if not identical, to interleukin 1 (IL-1). Both ETAF and IL-1 have been demonstrated to be chemotactic for neutrophils and mononuclear cells in vitro. In order to demonstrate that this activity has physiological relevance we have used a simple in vivo model. The present study demonstrates that injection of high-titer ETAF or purified recombinant murine IL-1 into the mouse footpad results in an influx of neutrophils into the site with peak accumulation at 4 h. Footpad swelling also occurs with a time course roughly paralleling that of the neutrophil accumulation. Injection of control proteins failed to reproduce this phenomenon. Margination of neutrophils within blood vessels was seen within 1 h of injection of ETAF or IL-1, followed by entry into the stroma by 4 h. This suggests that chemotactic activity and not merely increased adherence or inhibition of migration is occurring. 5-10 d of daily, subcutaneous injection of ETAF on the mouse flank resulted in an infiltrate of neutrophils, and to a lesser degree, mononuclear cells in association with epidermal hyperplasia, subcutaneous fibrosis, and focal muscle necrosis in the panniculus carnosus. These findings were not seen in control sites injected with media. These findings provide direct in vivo experimental evidence suggesting a physiologic role for ETAF/IL-1 in local inflammation.

Tumor antigen presentation by epidermal antigen-presenting cells in the mouse : modulation by granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and ultraviolet radiation

I‐A+ epidermal antigen‐presenting cells (APCs, Langerhans cells) have been shown to present tumor‐associated antigens (TAAs) and to induce tumor immunity in vivo. This study examined the effects of ultraviolet radiation (UVR) and the cytokines granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and tumor necrosis factor α (TNF‐α) on the ability of epidermal cells (ECs) to induce or to elicit immunity against the murine spindle cell tumor S1509a. Naive syngeneic mice were immunized three times at weekly intervals with ECs that had been cultured in GM‐CSF for 18 h and then pulsed with TAA derived from S1509a. This resulted in protective immunity against subsequent tumor challenge, providing a model to study the conditions required for sensitization against TAAs by epidermal APCs. Culture of ECs in GM‐CSF was required for induction of significant protective tumor immunity, and UV irradiation or incubation in TNF‐α for 2 h after GM‐CSF incubation abrogated the immunostimulatory effect of GM‐CSF. However, unlike UVR, TNF‐α did not significantly inhibit the induction of immunity when ECs were exposed to TNF‐α before overnight incubation in GM‐CSF, together with GM‐CSF, or after pulsing with TAA, and anti‐TNF‐α antibody treatment did not abrogate the effects of UVR on this system. Furthermore, TNF‐α incubation of ECs augmented their ability to elicit delayed‐type hypersensitivity (DTH) and also enhanced elicitation of DTH by GM‐CSF–cultured ECs, whereas UV‐irradiation reduced it in a dose‐dependent fashion. Taken together, these results demonstrate that GM‐CSF, TNF‐α, and UVR are significant regulators of tumor antigen presentation by epidermal APCs and that the effects of the cytokines examined differ with regard to induction or elicitation of immunity.

Neuropeptides and neuroendocrine hormones in ultraviolet radiation-induced immunosuppression

Exposure of the skin to ultraviolet radiation (UVR) can lead to deleterious effects such as sunburn, photoaging, and the development of skin cancer. UVR has also been shown to reduce local and systemic immune responses in humans and animals. In the recent past it has become clear that neuropeptides mediate some of the effects of UVR-induced immunosuppression. Among the neuropeptides released from cutaneous nerves after exposure to UVR, calcitonin gene-related peptide (CGRP) has been examined most extensively. It appears to lead to a reduction of contact hypersensitivity by inducing mast cells to degranulate and thus release tumor necrosis factor alpha (TNF-alpha) and, most likely, interleukin (IL)-10. Nitric oxide, which is coreleased with CGRP, seems to also play a role in immunosuppression through a yet undiscovered mechanism of action, while substance P may have counterregulatory effects. New evidence suggests that the release of neuropeptides from cutaneous sensory c-fibers after UVR is induced by keratinocyte-derived nerve growth factor. UVR can also induce epidermal and some dermal cells, such as melanocytes, keratinocytes, and dermal microvascular epithelial cells, to produce proopiomelanocortin (POMC) and its derivatives. The POMC product alpha-melanocyte-stimulating hormone (alpha-MSH) has been implicated in suppression of contact hypersensitivity and induction of hapten-specific tolerance, most likely by inducing keratinocytes and monocytes to produce the anti-inflammatory cytokine IL-10. Other POMC derivatives have not yet been investigated with regard to a possible role in UVR-induced effects on immunity.

RNA as a tumor vaccine: a review of the literature

Abstract: Many approaches have been attempted to harness the host immune system to act against malignant tumors. These have included animal and clinical trials with agents to non‐specifically boost immunity, factors to augment specific immunity, transfer of lymphokine‐activated killer cells and transfer of expanded populations of tumor‐infiltrating lymphocytes. Therapeutic vaccination strategies have been employed using tumor extracts, purified tumor antigens, recombinant peptide tumor antigens and specific DNA sequences coding for a tumor antigen (genetic vaccination) both through direct administration to the host and by administration of antigen presenting cells exposed to these materials ex vivo. Recently, the use of RNA has been proposed for use in tumor vaccination protocols. The use of RNA has several potential advantages. Since total cellular RNA or mRNA can be utilized, it is not necessary to know the molecular nature of the putative tumor antigen(s). RNA can be effectively amplified; thus, unlike tumor‐extract vaccines, only a small amount of tumor is needed to prepare the material for vaccination. Also, unlike DNA‐based vaccines, there is little danger of incorporation of RNA sequences into the host genome. The possible utility of RNA‐based vaccines for tumor immunotherapy should be further explored to determine whether such approaches are clinically useful.