Irene Dougherty

Counterregulation between the FcεRI Pathway and Antiviral Responses in Human Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) play essential roles in directing immune responses. These cells may be particularly important in determining the nature of immune responses to viral infections in patients with allergic asthma as well those with other atopic diseases. The purposes of this study were 1) to compare the functional capacity of pDCs in patients with one type of allergic disorder, allergic asthma, and controls; 2) to determine whether IgE cross-linking affects antiviral responses of influenza-exposed pDCs; and 3) to determine whether evidence of counterregulation of FcεRIα and IFN-α pathways exists in these cells. pDC function was assessed in a subset of asthma patients and in controls by measuring IFN-α production after exposure of purified pDCs to influenza viruses. FcεRIα expression on pDCs was determined by flow cytometry in blood samples from patients with allergic asthma and controls. pDCs from patients with asthma secreted significantly less IFN-α upon exposure to influenza A (572 versus 2815; p = 0.03), and secretion was inversely correlated with serum IgE levels. Moreover, IgE cross-linking prior to viral challenge resulted in 1) abrogation of the influenza-induced pDC IFN-α response; 2) diminished influenza and gardiquimod-induced TLR-7 upregulation in pDCs; and 3) interruption of influenza-induced upregulation of pDC maturation/costimulatory molecules. In addition, exposure to influenza and gardiquimod resulted in upregulation of TLR-7, with concomitant downregulation of FcεRIα expression in pDCs. These data suggest that counterregulation of FcεRI and TLR-7 pathways exists in pDCs, and that IgE cross-linking impairs pDC antiviral responses.

Syndecan-4 Mediates the Coinhibitory Function of DC-HIL on T Cell Activation

Receptor-ligand interactions between APCs and T cells determine whether stimulation of the latter leads to activation or inhibition. Previously, we showed that dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) on APC can inhibit T cell activation by binding an unknown ligand expressed on activated T cells. Because DC-HIL binds heparin/heparan sulfate and heparin blocks the inhibitory function of DC-HIL, we hypothesized that a heparin/heparan sulfate proteoglycan on activated T cells is the relevant ligand. Screening assays revealed that syndecan-4 (SD-4) is the sole heparan sulfate proteoglycan immunoprecipitated by DC-HIL from extracts of activated T cells and that blocking SD-4 abrogates binding of DC-HIL to activated T cells. Moreover, cell-bound SD-4 ligated by DC-HIL or cross-linked by anti-SD-4 Ab attenuated anti-CD3 responses, whereas knocked-down SD-4 expression led to enhanced T cell response to APC. Blockade of endogenous SD-4 using specific Ab or soluble SD-4 receptor led to augmented T cell reactions to syngeneic and allogeneic stimulation in vitro and exacerbated contact hypersensitivity responses in vivo. We conclude that SD-4 is the T cell ligand through which DC-HIL mediates its negative coregulatory function.

Genomic scale analysis of the human keratinocyte response to broad-band ultraviolet-B irradiation

Ultraviolet B (UVB) radiation is an important inducer of many biologic changes in skin, of which keratinocytes are a key target. To gain better insight into changes in gene expression generated in the early phase after UVB exposure, we used complementary RNA (cRNA) microarray hybridization to compare differences in mRNA expression of UVB‐irradiated (single dose of 100 J/m2 broad‐band UVB) and sham‐irradiated primary cultured human keratinocytes. Six hours after irradiation, total RNA was isolated from keratinocytes, and cRNA was synthesized and hybridized to a GeneChip expression array (Affymetrix) consisting of 6800 genes. Based on a threshold of > twofold change, 187 genes (2.8%) were designated to be the most UVB‐responsive. Surprisingly, none of these genes had been shown previously to be modulated by UVB. Conversely, several genes in the microarray that had been reported previously to be UVB‐ responsive by other methods showed less (< twofold) or no change. Northern blotting of seven differentially modulated genes produced results similar to those derived from microarray technology, thereby validating the accuracy of screening. Clustering based on known or likely functions indicated that among 88 upregulated genes, nine encode for cytochrome c subunits, six for ribosomal proteins, and two for regulators of apoptosis. By contrast, many of the 99 downregulated genes are involved in transcription, differentiation and transport. These findings indicate that keratinocytes respond to a single low dose of broad‐band UVB irradiation by enhancing processes involved in energy production and translation, while suppressing those related to transcription, differentiation and transport.

Inhibition of the elicitation phase of contact hypersensitivity by thymidine dinucleotides is in part mediated by increased expression of interleukin-10 in human keratinocytes

Abstract: The production of immunomodulatory cytokines such as interleukin‐10 (IL‐10) from keratinocytes and other target cells in the skin plays a crucial role in UV‐induced immunosuppression. Substantial evidence supports an association between DNA damage and immunomodulation. It is also known that small DNA fragments such as thymidine dinucleotides (pTpT) can mimic several UV‐induced effects, including inhibition of the induction phase of the contact hypersensitivity response and up‐regulation of tumor necrosis factor‐alpha (TNF‐α). To determine whether pTpT also induces IL‐10 secretion by keratinocytes, and by inference whether IL‐10 production after UV irradiation is a response to DNA damage, we compared the effects of pTpT with those of UV irradiation on primary human keratinocyte cultures. Subconfluent cultures of primary human keratinocytes were treated either with 10 µM or 100 µM pTpT or diluent alone, or exposed to solar‐simulated light (100 J/m2 of UVB) or sham irradiated. An increase in IL‐10 mRNA expression was observed 6–24 h after irradiation and at 24–48 h after treatment with pTpT. Detection of secreted IL‐10 protein coincided with up‐regulation of IL‐10 gene expression at 48 and 72 h as determined by ELISA. Conditioned media from human keratinocytes treated with pTpT, like that from irradiated cells, significantly inhibited lymphocyte proliferation in the allogeneic‐mixed lymphocyte reaction (MLR) assay. To determine whether pTpT mimics the suppressive influence of UVB on the elicitation phase of contact hypersensitivity, believed to result largely from IL‐10 release, we compared the effects of topical application of pTpT with those of UVB irradiation on C57Bl/6 mice sensitized with dinitrofluorobenzene. Sensitized mice treated with pTpT or UVB irradiation showed markedly suppressed elicitation of ear‐swelling responses. These results demonstrate that increased keratinocyte IL‐10 mRNA level and IL‐10 protein release are among the effects of pTpT and support the hypothesis that pTpT treatment triggers many of the biologic effects of UV irradiation by mimicking UV‐induced DNA damage. Finally, regardless of mechanism, the data suggest that topical treatment with pTpT may provide a novel means of suppressing contact hypersensitivity or other lymphocyte‐mediated reactions in skin.

Activation of HIV in human skin by ultraviolet B radiation and its inhibition by NFκB blocking agents

Abstract To determine whether ultraviolet B (UVB) irradiation leads to activation of HIV in human skin, we conducted prospective and controlled studies in two academic medical centers in Texas from July 1995 to April 1999. HIV-positive patients with UV-treatable skin diseases were enrolled at each center, 18 subjects at one and 16 at the other. In one center, specimens from lesional and nonlesional skin biopsies were taken before and after sham- or UVB-irradiation administered in vivo or in vitro. In the other center, UVB phototherapy was administered three times weekly and specimens from skin biopsies were taken before and after 2 weeks (six treatments). Cutaneous HIV load was assessed using reverse transcriptase-polymerase chain reaction and reverse transcriptase-polymerase chain reaction in situ hybridization. UVB irradiation led to a 6–10-fold increase in the number of HIV in skin. To ascertain a role for nuclear factor kappa B (NFκB) in UVB-inducible HIV activation, two types of blockers, NFκB oligonucleotide decoy and sodium salicylate, were tested; each inhibited UVB-inducible HIV activation in skin partially. We conclude that UVB irradiation leads to increased numbers of HIV in human skin via processes that include release of cytoplasmic NFκB.

Interleukin-15 expression by human endothelial cells: up-regulation by ultraviolet B and psoralen plus ultraviolet A treatment.

The purposes of the present study were to determine whether endothelial cells express IL‐15 and to evaluate effects of ultraviolet B (UVB) and 8‐methoxypsoralens plus UVA (PUVA) on such expression. Cultured human endothelial cells derived from dermis or umbilical veins were subjected to reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunoblot analyses for the detection of IL‐15 mRNA and protein, respectively. Both dermal and umbilical vein endothelial cells were shown to express IL‐15 mRNA and protein, and these markers were upregulated following UVB or PUVA treatment (but not by UVA or 8‐methoxypsoralens alone). Also using RT‐PCR, dermal and umbilical vein endothelial cells were shown to express IL‐2Rγc mRNA. These results expand the sources of IL‐15 in skin to include keratinocytes, dermal fibroblasts, and now endothelial cells. That IL‐15 from all three skin cells can be upregulated by UV treatment suggests a role for this cytokine in photosensitive disorders. Finally, the possibility of an autocrine effect of IL‐15 on endothelial cells is raised by the expression of IL‐2Rγc in these cells.

Modified peanut oral immunotherapy protocol safely and effectively induces desensitization

Peanut oral immunotherapy (OIT) has been shown to effectively induce desensitization in the majority of peanut-allergic individuals who receive therapy. However, questions exist regarding its safety and the optimal dose of peanut protein to administer. Studied maintenance doses range from 800 mg daily to 4000 mg daily. Side effects, which are typically mild, are most often seen during the build-up phase of therapy; therefore, we hypothesized that minimizing time to maintenance dosing may be favorable for the safety of individuals over the course of the therapy. We designed an unblinded peanut OIT pilot study that shortened time to maintenance by incorporating (1) a modified entry dose protocol and (2) a 2000 mg maintenance dose. Desensitization endpoints comparable to previously published protocols utilizing a 4000 mg maintenance dose were used for comparison. Inclusion and exclusion criteria are outlined in Table E1 (available in this article’s Online Repository at www.jaciinpractice.org) and required objective symptoms of allergic reactivity within 60 minutes of peanut ingestion during a double-blind, placebo-controlled food challenge (DBPCFC) to 2000 mg of peanut protein. Dose initiation was performed as outlined in Table I, utilizing lightly roasted peanut flour (partially defatted 12% fat; Golden Peanut Co., Alpharetta, Ga; 2 g flour 1⁄4 1 g peanut protein). A modified entry dose was based on the threshold dose of reactivity. Build-up dosing occurred approximately every 2 weeks. After dose initiation, doses were increased as previously described. A 5000 mg DBPCFC was performed after approximately 4 months of maintenance dosing (median 105 days, range 83 to 181 days). Subjects were instructed to resume 2000 mg daily dosing after the DBPCFC. Titrated skin prick tests (SPTs) were performed every 6 months with peanut extract (Greer Laboratories, Lenoir, NC). Peanutspecific IgE and IgG4 levels were measured using the ImmunoCAP 100 instrument (Phadia AB, Uppsala, Sweden) according to the manufacturer’s instructions. Approval for this study was obtained through the University of Texas Southwestern Institutional Review Board. Differences in the values over time compared with baseline were analyzed by using the Wilcoxon rank-sum tests (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, Calif) on matched data. P values <.05 were considered significant. Twelve subjects were screened and 11 met entry criteria (see Table E2 in this article’s Online Repository at www.jaciinpractice.org). Of the 11 subjects, 9 were able to safely ingest the 2000 mg daily maintenance dose. Details regarding the 2 subjects unable to achieve maintenance are included in Table E2. The median time to maintenance was 41 weeks (28-48 weeks) (Table E2). The most common reason to delay up-dosing was related to convenience (eg, inability to miss school, study staff vacation, etc.). Adjusting the time to maintenance with removal of convenience factors yields a median time to maintenance of 36 weeks (26-45 weeks). All 9 subjects who achieved maintenance dosing passed the 5000 mg peanut DBPCFC (Figure 1). Three subjects experienced transient symptoms during the challenge, which were not deemed significant enough to discontinue the challenge. From entry to the 5000 mg DBPCFC, 3265 total doses were administered. Only 7.9% of doses (264/3265) were associated with a reported side effect. All side effects were mild except for 2 severe reactions (see Table E3 in this article’s Online Repository at www.jaci-inpractice.org). The majority of reactions, 94% (249/ 264), occurred during the build-up phase. The most common complaints during build-up included skin reactions, followed by throat clearing and sneezing (see Figure E1 in this article’s Online Repository at www.jaci-inpractice.org). The skin prick test wheal diameter decreased significantly in all subjects after 6 months of therapy (see Figure E2A in this article’s Online Repository at www.jaci-inpractice.org). Peanutspecific IgE, IgG4, and IgE/IgG4 changed significantly after 6 weeks of therapy (see Figure E2B-D in this article’s Online Repository at www.jaci-inpractice.org), similar to findings from investigators using other protocols. Secondary to our modified entry dose protocol with a lower maintenance dose, our subjects were able to achieve maintenance dosing in a shorter time frame, and with comparable levels of desensitization and similar effects on the immune response as reported by previous investigators. Making head-to-head comparisons between existing peanut OIT studies is difficult secondary to the varied approaches investigators have taken. Begin et al recently reported successful acquisition of maintenance dosing by an average of 18 weeks with 4000 mg dosing per allergen for multiple allergen OIT; however, omalizumab is given adjunctively during up-dosing. Our up-dosing regimen was longer, but we did not use omalizumab. Our reaction rate was similar (5.3% [Begin] vs 7.9% [Bird]) without the protective benefits of omalizumab. We have now shown that 9/9 children who received a 2000 mg maintenance dose were able to pass a 5000 mg challenge with 6/9 (66%) having no symptoms during the challenge, and they were able to achieve maintenance dosing in a median time of 41 weeks. Because of differences in time receiving maintenance, total time on therapy, and differences in the amount of protein given during the desensitization challenge, we cannot directly compare our study with the recent study by Anagnostou et al. It

Adenovirus‐mediated blockade of lymphotoxin‐β inhibits the induction of contact sensitivity in mice

Abstract Lymphotoxin‐β is a newly recognized member of the tumor necrosis factor ligand family. Recent studies have suggested a role for this cytokine in delayed‐type hypersensitivity responses. To determine whether lymphotoxin‐β contributes to the development of contact sensitivity, we utilized an inhibitor protein that can effectively block binding of lymphotoxin‐β to its receptor. An adenoviral vector was created that encodes for a lymphotoxin‐β inhibitor protein consisting of the extracellular domain of the lymphotoxin‐β receptor fused to IgG heavy chain. Intravenous injection of the recombinant virus into BALB/c mice yielded plasma levels of inhibitor protein >500 μg that persisted for 1 week. Mice treated in this manner were compared with control animals injected with adenovirus encoding β‐galactosidase, with respect to their ability to mount contact sensitivity responses to epicutaneously applied dinitro‐fluorobenzene. Mice transduced with the lymphotoxin‐β inhibitor prior to the induction of contact sensitivity showed significantly suppressed ear swelling responses. By contrast, mice treated with the lymphotoxin‐β inhibitor prior to the elicitation of contact sensitivity showed no change in ear swelling responses in comparison to controls. These findings indicate that lymphotoxin‐β plays an important role in the afferent phase of the contact sensitivity response.