Visualization of atherosclerosis as detected by coronary artery calcium and carotid intima-media thickness reveals significant atherosclerosis in a cross-sectional study of psoriasis patients in a tertiary care center.

BACKGROUND: Psoriasis is a chronic inflammatory disease of the skin and joints that may also have systemic inflammatory effects, including the development of cardiovascular disease (CVD). Multiple epidemiologic studies have demonstrated increased rates of CVD in psoriasis patients, although a causal link has not been established. A growing body of evidence suggests that sub-clinical systemic inflammation may develop in psoriasis patients, even from a young age. We aimed to evaluate the prevalence of atherosclerosis and identify specific clinical risk factors associated with early vascular inflammation. METHODS: We conducted a cross-sectional study of a tertiary care cohort of psoriasis patients using coronary artery calcium (CAC) score and carotid intima-media thickness (CIMT) to detect atherosclerosis, along with high sensitivity C-reactive protein (hsCRP) to measure inflammation. Psoriasis patients and controls were recruited from our tertiary care dermatology clinic. Presence of atherosclerosis was defined using validated numeric values within CAC and CIMT imaging. Descriptive data comparing groups was analyzed using Welch's t test and Pearson Chi square tests. Logistic regression was used to analyze clinical factors associated with atherosclerosis, and linear regression to evaluate the relationship between psoriasis and hsCRP. RESULTS: 296 patients were enrolled, with 283 (207 psoriatic and 76 controls) having all data for the hsCRP and atherosclerosis analysis. Atherosclerosis was found in 67.6 % of psoriasis subjects versus 52.6 % of controls; Psoriasis patients were found to have a 2.67-fold higher odds of having atherosclerosis compared to controls [95 % CI (1.2, 5.92); p = 0.016], after adjusting for age, gender, race, BMI, smoking, HDL and hsCRP. In addition, a non-significant trend was found between HsCRP and psoriasis severity, as measured by PASI, PGA, or BSA, again after adjusting for confounders. CONCLUSIONS: A tertiary care cohort of psoriasis patients have a high prevalence of early atherosclerosis, increased hsCRP, and psoriasis remains a risk factor for the presence of atherosclerosis even after adjustment of key confounding clinical factors. Psoriasis may contribute to an accelerated systemic inflammatory cascade resulting in increased risk of CVD and CV events.

Does poor sleep quality affect skin ageing?

BACKGROUND: Sleep is important for growth and renewal of multiple physiological systems. The effects of chronic poor sleep quality on human skin function and visible signs of ageing have not been elucidated. AIM: To evaluate the effect of chronic poor sleep quality on measures of skin health and ageing. Self-perceived satisfaction with appearance was also assessed. METHODS: 60 healthy caucasian women, who were categorized as poor quality sleepers [Pittsburg Sleep Quality Index (PSQI) > 5, sleep duration ≤ 5 h] or good quality sleepers (PSQI ≤ 5, sleep duration 7-9 h). A validated clinical tool, SCINEXA(TM) , was used to assess intrinsic and extrinsic skin ageing. Dark under-eye circles were evaluated using standardized photos. Measurement of in vivo transepidermal water loss (TEWL) was used to assess recovery of the skin barrier after tape stripping. Subjects were exposed to simulated solar ultraviolet light, and recovery from erythema was monitored. Subjects also completed a questionnaire evaluating self-perception of attractiveness. RESULTS: Good sleepers had significantly lower intrinsic skin ageing scores by SCINEXA(TM) . At baseline, poor sleepers had significantly higher levels of TEWL. At 72 h after tape stripping, good sleepers had 30% greater barrier recovery compared with poor sleepers. At 24 h after exposure to ultraviolet light, good sleepers had significantly better recovery from erythema. Good sleepers also reported a significantly better perception of their appearance and physical attractiveness compared with poor sleepers. CONCLUSIONS: This study indicates that chronic poor sleep quality is associated with increased signs of intrinsic ageing, diminished skin barrier function and lower satisfaction with appearance.

Successful cutaneous delivery of the photosensitizer silicon phthalocyanine 4 for photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) has been shown to be effective in the treatment of malignancies of a variety of organ systems, including the lungs, bladder, gastrointestinal tract and skin. Cutaneous lesions serve as ideal targets of PDT because of the accessibility of the skin to light. To achieve optimum results, the photosensitizer must be delivered effectively into the target layers of the skin within a practical timeframe, via noninvasive methods. AIM: To determine whether topical application of a second-generation photosensitizer, silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2 (CH2)3 N(CH3)2)(OH)], results in effective penetration of the skin barrier. METHODS: Penetration of Pc 4 was evaluated using standard Franz-type vertical diffusion cell experiments on surrogate materials (silicone membranes) and laser-scanning confocal microscopy of normal skin biopsy samples from human volunteers. RESULTS: The Franz diffusion data indicate that Pc 4 formulated in an ethanol/propylene glycol solution (70/30%, v/v) can penetrate the membrane at a flux that is appreciable and relatively invariant. Using the same formulation, Pc 4 uptake could be detected in human skin via laser-scanning confocal microscopy. CONCLUSION: After topical application, Pc 4 is absorbed into the epidermis in as little as 1 h, and the absorption increased with increasing time and dose. Pc 4 can be effectively delivered into human skin via topical application. The data also suggest that the degree of penetration is time- and dose-dependent.

A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line.

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.

In this study we define the proliferative compartments of in vivo human epidermis, using specific antibodies related to cell differentiation (beta 1 and beta 4 integrins and K1/K10 differentiation keratins) and cell cycle (proliferating cell nuclear antigen [PCNA]) in combination with flow cytometric quantitation of the DNA content and optical characteristics of the cells. The beta 1 integrin (CD29) marked both of the potentially proliferative subsets in normal epidermis. One subset of normal epidermis is CD29+ K1/K10-, which was predominantly basal, and found to be comprised of slow cycling, small cells with primitive cytoplasmic organization. The vast majority (95.5%) of these cells were in a quiescent state (G0/early G1) as indicated by their lack of the cyclin, PCNA. The other proliferative subset of normal epidermis was CD29+ K1/K10+, which was suprabasal and occasional basal, highly proliferative, larger in size, and which exhibited a more complex cytoplasmic structure. Because early differentiation (K1/K10 expression) has begun in the CD29+ K1/K10+ subset, it is highly likely that they represent the proliferative population which is capable of transiently amplifying itself before terminal differentiation. Within lesional psoriatic epidermis, similar proliferative cell populations were present as in normal epidermis, and the hyperproliferative defect was localized to the beta 1 and beta 4 integrin+, K1/K10- populations, which in normal epidermis is basally located and quiescent with regard to cell cycle. In psoriatic epidermis, a six- to sevenfold increase in the number of cells in the S/G2+M phase of cell cycle was found among CD29+ K1/K10- cells (p < 0.05). Furthermore, all lesional K1/K10- cells showed high PCNA positivity, indicating that all these cells had been recently induced into cell cycle. By contrast, the proportion of cycling cells among lesional psoriatic CD29+ K1/K10+ keratinocytes was similar to normals. Anti-HLA-DR, CD45, and vimentin antibodies were used to concomitantly track the proliferative states of Langerhans cell, melanocyte, and infiltrating leukocyte populations. In normal epidermis, the cycling fractions (cells in S/G2/M phase) of these cells were similar to the CD29+K1/K10- keratinocytes, whereas in lesional epidermis their cycling pools were increased relative to normal, but not so much as the proliferative fractions of psoriatic CD29+ K1/K10- keratinocytes. These data demonstrate the use of simultaneous analysis of integrin expression, differentiation keratins, cyclin, cell cycle status, and optical characteristics of freshly isolated human epidermal cells. Such analysis allowed the physical identification and quantification of cy cling populations in normal human skin, and has enabled the precise location of the primary epidermal proliferative defect in psoriasis.

Activated complement component 3 (C3) is required for ultraviolet induction of immunosuppression and antigenic tolerance.

Complement component 3 (C3), a critical regulator of innate immunity, may also play a role in the regulation of cognate immunity, such as contact sensitivity responses. Because ultraviolet (UV) radiation also activates C3 in the skin, we determined whether the immunosuppressed state that results when a contact sensitizer is applied through UVB-exposed skin requires the presence and activation of C3. This question was addressed through the use of C3-deficient mice, blockade of C3 cleavage to C3b, and accelerated degradation of iC3b by soluble complement receptor 1 (sCR1). Both C3-modulated systems totally reversed the failure to induce a contact sensitivity response to dinitrofluorobenzene (DNFB) upon primary sensitization at the UV-exposed site, as well as immunologic tolerance to a second DNFB immunization through normal skin. Treatment with sCR1 reduced the infiltration of CD11b+ leukocytes into the epidermis and dermis of UV-irradiated skin but did not reverse the UV-induced depletion of epidermal class II MHC+CD11blo Langerhans cells. These data, taken together with previous results showing abrogation of locally induced UV immunosuppression by in vivo anti-CD11b treatment, suggest a novel mechanism by which ligation of the leukocyte beta2 integrin, CD11b, by iC3b molecules formed from C3 activation in UV-exposed skin, modifies cutaneous CD11b+ cells such that skin antigen-presenting cells are unable to sensitize in a primary immune response, but actively induce antigenic tolerance.

Malignant T cells in cutaneous T-cell lymphoma lesions contain decreased levels of the antiapoptotic protein Ku70.

BACKGROUND: Malignant T cells in primary cutaneous T-cell lymphoma (CTCL) are genetically unstable and exhibit prolonged lifespans potentially explained by dysregulation of apoptosis, yet are responsive to apoptosis-inducing therapies. The heterodimeric protein Ku70/80 is known to play a role in DNA repair (Ku70 and Ku80) and inhibition of apoptosis (Ku70 only). OBJECTIVES: To investigate the expression of Ku70/80 in CD3+ T cells derived from skin and blood in patients with CTCL and normal samples, as well as benign dermatoses. METHODS: Normal (n=10), CTCL (n=9) and benign dermatoses (n=13) skin samples were stained for confocal imaging of Ku70/80 and CD3 and analysed using imaging software. Circulating CD4+ T cells in normal and CTCL peripheral blood were analysed by flow cytometry and Western blot for Ku70/80 expression (n=6). RESULTS: Ku70 and Ku80 were significantly diminished in T cells of CTCL lesions relative to T cells of control skin. Decreased T-cell Ku70 expression was not a feature of the benign dermatoses psoriasis and contact dermatitis, suggesting that loss of Ku70/80 in CTCL is not simply the result of cutaneous inflammation. Reduced Ku70 was also noted in circulating CD4+ T cells in patients with CTCL with peripheral blood involvement. CONCLUSIONS: Deficient expression or lack of Ku70/80 may result in genomic instability and play a role in tumorigenesis, as well as account for the increased susceptibility of malignant T cells to apoptosis-inducing treatment modalities in the setting of intrinsic resistance to apoptosis.

Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes.

Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis.

Rapamycin (sirolimus) inhibits proliferating cell nuclear antigen expression and blocks cell cycle in the G1 phase in human keratinocyte stem cells.

Because the immunosuppressant rapamycin (sirolimus) blocks T cell proliferation in G1 phase, it has been proposed as a potential treatment for psoriasis, a skin disease characterized by T cell activation and keratinocyte stem cell hyperproliferation. To determine another potentially important mechanism through which rapamycin can act as an antipsoriatic agent, we tested its direct effect on keratinocyte stem cell proliferation in vitro as well as in vivo. In vivo cell cycle quiescent (G0 phase) stem cell keratinocytes in primary culture sequentially express de novo cyclin D1 and proliferating cell nuclear antigen (PCNA), prior to S phase entry, and upregulate beta1 integrin. Rapamycin inhibited the growth of keratinocytes that were leaving quiescence as well as those already in cell cycle without affecting cell viability. Although beta1 integrin(bright) expression was not affected, the number of beta1 integrin(bright) cells entering S/G2/M was significantly lowered by rapamycin. Cells treated with rapamycin exhibited decreased PCNA expression while cyclin D1 expression, which precedes PCNA expression in the cell cycle, was not affected. We found similar effects on stem cell keratinocytes in patients with psoriasis treated systemically with rapamycin. Because PCNA is required for cell cycle progression from G1 to S phase, our data indicate that inhibition of PCNA protein synthesis may be an important regulatory element in the ability of rapamycin to exert a G1 block.

Interleukin-1 receptor antagonist in normal and psoriatic epidermis.

The objective of these studies was to characterize the IL-1 inhibitory activity present in normal and psoriatic epidermis from clinically stable lesions. Fractionation of normal epidermal cytosol on a molecular sizing column failed to reveal the presence of IL-1 inhibitory bioactivity. However, specific ELISAs indicated that both the IL-1 receptor antagonist (IL-1ra) and IL-1 alpha were present in overlapping peaks. Further fractionation of the normal epidermal cytosol by anion exchange chromatography separated these two molecules, revealing the IL-1 inhibitory bioactivity of the IL-1ra molecule. Similar studies on psoriatic epidermal cytosol indicated the presence of IL-1 inhibitory bioactivity and IL-1ra protein. The IL-1 inhibitory bioactivity of both normal and psoriatic cytosol was neutralized by a mAb specific for IL-1ra. The ratio of IL-1ra to IL-1 alpha proteins was significantly increased in involved psoriatic skin compared with normal skin. By Western blot analysis this IL-1ra was approximately 20 kD, slightly larger than monocyte-derived IL-1ra and equivalent to an intracellular variant of IL-1ra expressed by keratinocytes. Polymerase chain reaction indicated the presence of mRNA for both forms of IL-1ra in normal epidermis, with both forms increased in psoriatic-involved skin. Immunofluorescence studies revealed the IL-1ra protein to be concentrated in the stratum granulosum of normal skin and in the basal-midbasal layers of psoriatic epidermis. These results suggest that the balance between intracellular IL-1ra and IL-1 alpha may be an important influence on keratinocyte growth and/or differentiation, as well as on the inflammatory potential of IL-1 in injured skin.

Regulatory T cells in psoriasis.

Psoriasis is a chronic autoimmune disease in which T lymphocytes are thought to be central in the pathogenesis. Recently, a T cell subset population was identified, whose role is to suppress inflammatory responses triggered by T effector cells. T cells in this new population are referred to as T regulatory cells. We studied their number and activity in psoriatic lesions and found that they are both numerically and functionally deficient in their ability to suppress the abnormally persistent psoriatic immune response. This deficiency may shed more light on the complex pathophysiology of psoriasis.

Protein synthesis initiation factors from chicken erythroblasts.

Two factors (IF-I and IF-II) necessary for the initiation of protein synthesis have been partially purified from a 0.5 M KC1 wash of chicken erythroblast polysomes. IF-I mediates the binding of the initiator tRNA and GTP to a 40 S ribosomal subunit, resulting in the formation of a 44 S initiation intermediate. In the presence of IF-II and a suitable RNA template, the 44 S initiation intermediate combines with a 60 S ribosomal subunit to form a functional 80 S initiation complex. The methionyl moiety of the initiator tRNA in the 80 S initiation complex is able to react with puromycin to form methionylpuromycin.

The key role of aquaporin 3 and aquaporin 10 in the pathogenesis of pompholyx.

Pompholyx remains a chronic skin affliction without a compelling pathophysiological explanation. The disease is characterized by the sudden onset of vesicles exclusively in the palms and soles which generally resolves. However, the disease may progress and the vesicles may expand and fuse; with chronicity there is deep fissuring. Multiple therapeutic approaches are available, but the disease is often resistant to conventional treatments. Currently, oral alitretinoin is used for patients with resistant chronic disease; however, this therapy is only approved for use in the UK, Europe and Canada. In this paper we wish to put forward a hypothesis: exposure to water and the subsequent steep osmotic gradient imbalance are key factors driving skin dehydration seen in pompholyx patients once the disease becomes chronic. The mechanistic explanation for the epidermal fissuring might lie in the over-expression across the mid and upper epidermis, including the stratum corneum, of two water/glycerol channel proteins aquaporin 3 and aquaporin 10, expressed in the keratinocytes of afflicted pompholyx patients. The over-expression of these two aquaporins may bridge the abundantly hydrated dermis and basal epidermis to the outer environment allowing cutaneous water and glycerol to flow outward. The beneficial effects reported in alitretinoin-treated patients with chronic hand eczemas may be due potential regulation of aquaporin 3 and aquaporin 10 by alitretinoin.

Atopic dermatitis: recent trends in pathogenesis and therapy.

Emerging concepts in the areas related to the pathogenesis and treatment of atopic dermatitis are reviewed. In particular, recent findings have revealed several key steps in the maintenance of a vicious circle of spongiotic dermatitis associated with elevated T-lymphocyte activation, hyperstimulatory Langerhans cells, defective cell-mediated immunity, and B-cell IgE overproduction. The discovery of specific IgE-binding structures on Langerhans cells provides a mechanism for Langerhans cells to capture and present IgE-targeted allergens to allergen-specific T cells. Furthermore, certain microbial allergens that tend to preferentially elicit IgE-type responses also elicit a T-cell response dominated by the IgE-inducing lymphokine interleukin 4. Repeated stimulation by activated Langerhans cells appears to induce just such a response. Abnormal biochemical responsiveness and mediator release by AD monocytes, mast cells, and eosinophils also participate in the sustainment or initiation of such a vicious circle, and contribute directly to the dermatitis as well. Developments in the areas of neuropeptides, genetics, microbial superantigens, and cytokine networks in the skin also appear to have promise in providing a rational link between immune defects and the inflammatory events in AD. Conventional therapy remains the mainstay of atopic dermatitis management; however, new therapies based upon the above concepts are being tested in clinical trials. Although the difficulty of objectively grading AD lesional activity and the high placebo response of AD patients hampers the interpretation of many reports, several types of approaches are coming into focus. The effectiveness of cyclosporin A, which targets T-cell activation and antigen presentation, indicates that additional agents with such activity should be effective, and verifies the criticality of these cells in AD pathogenesis. Therapy with biologic response modifiers, such as interferon gamma or thymopentin, is oriented toward normalization of imbalanced immune responsiveness, rather than direct suppression of the immune system. The mechanism of action of and toxicities of Chinese herbal mixtures require further investigation, but may reveal hitherto unconsidered avenues. Other recent therapeutic trials have focused on reduction of trigger factors, such as house dust mite exposure, foods, and the abnormal epidermal lipid barrier to irritation.

Cutaneous dermal Ia+ cells are capable of initiating delayed type hypersensitivity responses.

The presence of Langerhans cells (LC) within the epidermis has been shown to be critical for inducing T-cell-mediated immune responses in the skin. The purpose of this study was to assess whether cells in the dermis can initiate T-cell-mediated delayed-type hypersensitivity responses in vivo. Initially, back skins from C3H mice were trypsinized to remove the epidermis. The dermis was enzymatically dispersed and filtered to obtain a cell suspension. However, dermal cells exposed to trypsin were contaminated with numerous disaggregated hair follicles. These hair follicles contained Ia+ cells (presumably LC), and upon haptenation in vitro with trinitrophenyl, initiated contact hypersensitivity reactions in vivo. We therefore used dispase in place of trypsin to prevent follicular disaggregation and to allow preparation of dermal cell suspensions free of hair follicles. These hair follicle-free dermal cells were haptenated with trinitrophenyl and injected intradermally. Elicitation of contact hypersensitivity by epicutaneous painting 6 d later revealed the mean +/- SEM incremental ear-swelling response to be 53 +/- 8 mm X 10(-3). In contrast, mice sensitized by injection with dermal cells depleted of Ia+ cells demonstrated only 10 +/- 1 mm X 10(-3) of ear swelling. Thus, like dendritic LC of the epidermis, perivascular dendritic Ia+ cells of the dermis are capable of initiating T-cell-mediated contact hypersensitivity in vivo and may be highly relevant for presentation of antigen to T cells trafficking through the dermis.

Differential extracellular signaling via Fc gamma R and FMLP in functionally distinct antigen-presenting cell subsets: ultraviolet-induced epidermal macrophages versus Langerhans cells.

Sunburned skin is characterized by expanded numbers of macrophages (ultraviolet [UV]-MPH), and these UV-MPH differ from Langerhans cells (LC) in their abilities to initiate T-cell-mediated immune reactions. UV-MPH and LC may themselves be differentially responsive to the surrounding milieu, which may in turn modulate their immunoregulatory activity. We asked whether immunologic signal responsiveness, as assessed by cytosolic calcium mobilization, differed among normal human LC, UV-MPH, and normal blood monocytes. LC from normal skin and UV-MPH from UV-exposed skin were distinguished from keratinocytes in epidermal cell suspensions by labeling with anti-HLA-DR. Intracellular calcium content was monitored in real time with the calcium indicator, indo-1, after cross-linking Fc gamma RI, Fc gamma RII, CD11b, CD11c, or CD18 molecules, or addition of interleukin-1 alpha, IL-1 beta, interferon-gamma, bradykinin, substance P, or FMLP. Using flow cytometric analysis of cell suspensions, UV-MPH and blood monocytes were triggered by cross-linking Fc gamma RII (flux of 6.05 and 12.2, respectively). UV-MPH could also be triggered by Fc gamma RI crosslinking and FMLP (flux of 6.41 and 15.54, respectively). By contrast, none of these inflammatory stimuli could cause cytosolic calcium mobilization in normal LC (Flux of -0.2 by FcRII, and 0.18 by FMLP). Because LC calcium flux may be dependent upon extracellular attachments, LC were anchored onto fibronectin-coated coverslips and then their Fc gamma RII was crosslinked in a continuous flow chamber. However, image analysis also failed to detect calcium flux. Neither population responded to interleukin-1, interferon-gamma, bradykinin, substance P, or beta 2 integrin crosslinking. These results indicate that blood monocytes and infiltrating macrophages differ substantially from LC in their responses to immune complexes and chemoattractants. Differential responsiveness to the inflammatory milieu may influence the antigen presenting or effector capabilities of these populations.

Temporal correlation between UV radiation locally-inducible tolerance and the sequential appearance of dermal, then epidermal, class II MHC+CD11b+ monocytic/macrophagic cells.

We performed a time course study in order to define the in vivo relationship between the induction of active suppression of contact sensitization and the presence of various cells in ultraviolet-exposed dermis and epidermis implicated in locally inducible immune tolerance: class II major histocompatibility complex (MHC)+CD11b(lo)Gr-1- Langerhans cells (LC), class II MHC-CD45+CD3+ dendritic epidermal T cells, class II MHC+CD11b+Gr-1- monocytes or class II MHC+CD11b+Gr-1+ monocytic/macrophagic cells. Partial tolerance (50%) was first detectable 6 h after a single 72 mJ/cm2 ultraviolet B exposure and maximum tolerance at 48 h post-ultraviolet exposure. By flow cytometry, a low granularity LC subset had disappeared from the epidermis within 6 h after ultraviolet exposure, followed by a slower decrease in the high granularity Langerhans cells subset. Within the dermis at the 6-h time point, small numbers of infiltrating monocytic/macrophagic cells are already apparent. By 24 h post-ultraviolet exposure, at which time tolerance has increased to 70%, the infiltrating monocytic/macrophagic population had risen to 1.2% of the total dermal cell population and was observed for the first time in the epidermis along with other infiltrating leukocytes (i.e., polymorphonuclear leukocytes). By 48 h post-ultraviolet exposure, when a state of maximum tolerance is obtained, both constitutive epidermal and dermal antigen-presenting cell populations were at or near their nadir of depletion. The infiltrating monocyte/macrophage population, however, exhibited a dramatic increase in the epidermis at 48 and 72 h. Thus, the ability to locally induce a state of in vivo tolerance is closely associated with the expansion of class II MHC+CD11b+Gr-1+ and -monocytic/macrophagic cells in the dermis and epidermis.

Significantly increased occurrence of HLA-DQB1*0301 allele in patients with ocular cicatricial pemphigoid.

To determine the immunogenetic characteristics of patients with immune-mediated subepithelial blistering diseases of mucous membranes, we performed HLA-typing for the class II MHC gene DQB1*0301 allele using a direct method. Genomic DNA extracted from Caucasian patients was amplified by polymerase chain reaction using primer pairs specific for the DQB loci followed by Southern blotting with a peroxidase-conjugated sequence-specific oligonucleotide probe. Seventy-six percent (16/21) of patients with ocular mucosal disease (with or without oral mucosal and skin diseases) carried the DQB1*0301 allele; by contrast, only 33% (14/42) of race-, age-, and geography-matched normal individuals carried the DQB1*0301 allele (p < 0.005). The relative risk for ocular disease if DQB1*0301 allele is present is 6.4, similar to the relative risk of 8 for patients with ocular but no oral disease (pure ocular cicatricial pemphigoid, p < 0.025). In patients with oral mucosal disease (with or without ocular mucosal and skin diseases), 68% (15/22) carried the DQB1*0301 allele (p < 0.025). When patients with ocular disease were excluded, however, the increased occurrence of the DQB1*0301 allele in patients with oral disease was not statistically significant (64%, 7/11, p < 0.25). In patients with subepidermal blistering skin disease but no oral or ocular disease, there was no increase in the occurrence of the DQB1*0301 allele (38%, 5/13, p > 0.5). The significantly increased occurrence of the DQB1*0301 allele in patients with ocular mucosal disease may point to a distinct immunogenetic factor that predisposes patients to develop an ocular scarring process.