Biologic therapy improves psoriasis by decreasing the activity of monocytes and neutrophils

Therapy with monoclonal antibodies to tumor necrosis factor (TNF)‐α and the interleukin (IL)‐12/23 p40 subunit has significantly improved the clinical outcome of patients with psoriasis. These antibodies inhibit the effects of the target cytokines and thus the major concern during their use is the induction of excessive immunosuppression. Recent studies evaluating the long‐term efficacy and safety of biologic therapy in psoriasis have shown no significant appearance of serious adverse effects including infections and malignancies. However, the immunological consequence and the mechanism by which the blockade of a single cytokine by biologics can successfully control the activity of psoriasis remain unclear. In the current study, we investigated the effect of biologic therapy on cytokine production of various lymphocytes and on the activity of monocytes and neutrophils in psoriatic patients. Neutrophils, monocytes and T cells were purified from heparinized peripheral venous blood by Ficoll density gradient centrifugation, and γ‐interferon, TNF‐α and IL‐17 production from lymphocytes was measured by flow cytometer. The activation maker of neutrophils and the activated subsets of monocytes were also analyzed. Biologic therapy induced no significant changes in the cytokine production by lymphocytes from the skin and gut‐homing T cells. However, neutrophil activity and the ratio of activated monocyte population increased in severely psoriatic patients were normalized in psoriatic patients receiving biologic therapy. The present study showed that biologic therapy ameliorates clinical symptoms and controls the immune response in patients with psoriasis.     

Enhancement of monocyte chemotactic activity in the sera of psoriatic patients after heat treatment

The chemotactic activity for neutrophils and monocytes was examined in the sera of psoriatic patients and healthy controls using a Boyden chamber method. There were no significant differences in neutrophil chemotactic activity between the sera of psoriatic patients and healthy controls. However, the monocyte chemotactic activity of psoriatic serum was lower than that of control serum. Furthermore, there was a significant increase in the monocyte chemotactic activity of psoriatic sera after heat treatment (42°C, 1 hr). These results suggest that psoriatic serum has a defect of monocyte chemotaxis which can be corrected, in part, by heat treatment.     

In vivo effects of PUVA on lymphocyte function

Eighteen patients with active psoriasis were investigated for antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by monocytes and neutrophil leukocytes. Patients with extensive psoriatic lesions showed increased ADCC whereas patients with minimal psoriasis had normal monocyte and neutrophil function. After clinical remission the ADCC became normal. No stimulatory factors in psoriatic serum could be demonstrated. The increased monocyte and neutrophil cytotoxicity in severe psoriasis is not explained by altered cyclic nucleotide levels as cAMP and cGMP levels were normal in psoriatic monocytes and neutrophils showing both increased and normal ADCC. Our results indicate that increased ADCC is secondary to the psoriatic activity.     

Enhanced monocyte and neutrophil cytotoxicity and normal cyclic nucleotide levels in severe psoriasis

Eighteen patients with active psoriasis were investigated for antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by monocytes and neutrophil leukocytes. Patients with extensive psoriatic lesions showed increased ADCC whereas patients with minimal psoriasis had normal mono cyte and neutrophil function. After clinical remission the ADCC became normal. No stimulatory factors in psoriatic serum could be demonstrated. The increased monocyte and neutrophil cytotoxicity in severe psoriasis is not explained by altered cyclic nucleotide levels as cAMP and cGMP levels were normal in psoriatic monocytes and neutrophils showing both increased and normal ADCC. Our results indicate that increased ADCC is secondary to the psoriatic activity.     

Mononuclear cell versus monocyte chemotaxis in psoriasis

Chemotaxis under agarose of psoriatic mixed mononuclear cells (MNs) and pure monocytes against Zymosan treated sera (ZAS), fresh psoriatic serum, and fresh normal serum was studied and compared with that of normal cells. Purification of monocytes was achieved on microexudate coated BHK flasks. Psoriatic MNs were found to be more chemotactic against psoriatic serum (P.S.) than normal MNs against normal serum (N.S.). Psoriatic cells were chemotactically more active than normal ones against psoriatic and normal serum but not against ZAS. Mixed mononuclear cells showed definitely increased chemotaxis over that of pure monocytes. When culture supernatants with lymphocyte derived factor (LDCF) activity were mixed with normal pure monocytes, the chemotactic activity of the monocytes recovered. It is, therefore, proposed that the LDCF from in vivo stimulated psoriatic lymphocytes by antigenic or mitogenic substances of psoriatic plaque might be an essential factor for the increased chemotaxis of mixed psoriatic MNs compared to pure monocytes. This hypothesis also explains the increased chemoattracting capacity of psoriatic serum for psoriatic and normal MNs as due to the in vivo release of lymphokine.     

白介素8及其受体CXCR2在银屑病角质形成细胞中的表达

目的 观察白介素8（IL－8）及基受体CXCR2在银屑病角质形成细胞中的表达及在银屑病的临床及病理表现中的作用。方法 培养银屑病患者皮损处角质形成细胞，通过微孔小室实验检测其上清液的趋化功能，通过酶联免疫吸附法（ELISA）检测上清液中IL－8的表达，通过流式细胞仪分析银屑病患者皮损处角质形成细胞的趋化因子受体CXCR2的表达。结果 银屑病患者皮损处角质形成细胞分泌上清液对中性粒细胞的趋化能力明显强于正常对照组，其分泌的IL－8水平也高于正常人，皮损处角质形成细胞CXCR2的表达也明显强于正常对照组。结论 银屑病患者皮损局部表面出的角质形成细胞高度增殖与角质形成细胞高分泌、高表达具有促增殖作用的IL－8及其受体CXCR2有关，同时皮损局部大量的炎性细胞的浸润部分可能是由于角质形成细胞高分泌具有趋化能力的IL－8，它们可能在银屑病的发生、发展中起着重要作用。     

The enhanced monocyte and neutrophil chemotaxis in psoriasis is normalized after treatment with psoralens plus ultraviolet A and anthralin

The chemotactic activity of purified monocytes and neutrophils was studied in twenty-two psoriatic patients prior to the initiation of psoralens plus ultraviolet A (PUVA) or anthralin therapy and during the treatment. Patients with untreated, active psoriasis had increased chemotactic response of their monocytes and neutrophils to N-formyl-methionyl-leucyl-phenylalanine. Plasma from untreated psoriasis patients had an enhanced chemotactic activity toward neutrophils from persons without psoriasis. Treatment with PUVA or anthralin resulted in a progressive clearing of psoriatic lesions and in a significant depression of leukocyte chemotaxis 2 weeks after the start of the respective therapy. The chemotactic activity of both monocytes and neutrophils was normalized after 4 weeks of anthralin therapy and after 6 weeks of PUVA treatment. The chemotaxis-enhancing properties of plasma from psoriasis patients returned to normal values already after 2 weeks of treatment.     

Cyclosporin increases granulocyte/macrophage colony-stimulating factor (GM-CSF) activity and gene expression in murine keratinocytes.

Keratinocytes produce multiple cytokines in response to a variety of stimuli. The release of interleukin 1 (IL-1) from keratinocytes may be significant in initiation of cutaneous inflammation, and the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) is thought to be important in the regulation of antigen-presenting function by epidermal Langerhans cells. Because cyclosporin inhibits interleukin 2 release from T cells, it has been suggested that cyclosporin may function as an anti-inflammatory agent within the epidermis through inhibition of keratinocyte cytokine release. This investigation examined the direct effect of cyclosporin on the production of GM-CSF by murine keratinocytes and the keratinocyte cell line PAM 212. GM-CSF bioactivity increased in cell supernatants from keratinocytes exposed in vitro to 1 microgram/ml cyclosporin for up to 24 h. GM-CSF and IL-1 mRNA levels in keratinocytes cultured under similar conditions or in the presence of lipopolysaccharide also increased. The lack of inhibition of GM-CSF expression following cyclosporin treatment is consistent with recent observations in T cells and is opposite to the effect of cyclosporin on interleukin 2.     

Neutrophil chemotaxis in psoriasis.

Neutrophil chemotaxis was assessed in 69 psoriatic patients and 37 healthy human subjects. It was found to be significantly enhanced in 52 untreated patients. In 20 patients treated with an orally-administered phosphodiesterase inhibitor, Diphylline, neutrophil chemotaxis was normal. The enhanced chemotactic response of neutrophils from untreated patients with minimal skin lesions was at least equal to the response of those from patients with extensive skin lesions. Preincubation of normal human leukocytes with plasma derived from patients with widespread lesions markedly reduced their chemotactic activity. Plasma derived from patients with extensive skin lesions exhibited marked chemoattracting properties in comparison with plasma from healthy subjects. It is postulated that the basic intrinsic abnormality of neutrophil function in psoriasis could be caused by a decreased cyclic AMP/cyclic GMP ratio, similar to the decreased cyclic AMP/cyclic GMP ratio found in the lesional epidermis of this disease. Plasma factors which influence chemotaxis in psoriasis are related to the extent of the eruption and their effect is contrary to the effect of the basic intrinsic abnormality of psoriatic neutrophils.     

Immunomodulating cytokines in atopic dermatitis and psoriasis: production of tumour necrosis factor and lymphotoxin by mononuclear cells in vitro

The immunomodulating cytokines, tumour necrosis factor/cachectin (TNF) and lymphotoxin (LT) are thought to play an essential role as mediators of inflammatory reactions. To evaluate the role of TNF and LT in atopic dermatitis (AD) and psoriasis, we investigated their production by mononuclear cells (MNC) in vitro. The 24-h supernatants of lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-stimulated and unstimulated MNC from 26 patients with AD and 20 with psoriasis and from 17 non-atopic healthy controls were tested for the concentrations of TNF and LT using an ELISA technique. In patients with AD, TNF levels were significantly decreased in the supernatant of PHA-stimulated (P less than or equal to 0.005) and LPS-stimulated (P less than or equal to 0.02) MNC in comparison to controls. There was no significant difference in TNF production between psoriatic patients and the control group. Release of LT in the supernatant of PHA-stimulated MNC by patients and controls did not differ significantly. There was no significant spontaneous production of TNF and LT by MNC of patients and controls. These studies indicate that different immunomodulating mechanisms are responsible for triggering the inflammatory response in AD and psoriasis.     

Human leukocyte elastase and cathepsin G are specific inhibitors of C5a-dependent neutrophil enzyme release and chemotaxis

Circulating human neutrophils from patients with severe inflammatory disorders such as erysipelas and sepsis are specifically desensitized to complement factor C5a stimulation but not to stimulation with other stimuli like N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), or platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). In this study, we raised the question whether factors released from polymorphonuclear leukocytes (PMNs) can specifically down-regulate C5a-dependent neutrophil functions. When neutrophils were preincubated with either neutrophil lysates or neutrophil degranulation supernatants, a complete inhibition of C5a-stimulated beta-glucuronidase release and chemotaxis could be observed, whereas FMLP-, IL-8-, LTB4- or PAF-dependent functions were not affected. Serine protease inhibitors like phenylmethylsulfonyl fluoride, antileukoprotease, or elafin abolished this effect. High-performance liquid chromatography of neutrophil degranulation supernatants revealed pronounced inhibition of C5a-dependent neutrophil functions in fractions exerting elastase or cathepsin G activity, but not in fractions exerting proteinase 3 activity. Using purified human leukocyte elastase (HLE), C5a responses like intracellular calcium influx, beta-glucuronidase release, and chemotaxis were also specifically inhibited. Our experiments show that the release of HLE or cathepsin G from neutrophils specifically down-regulates the responsiveness of neutrophils to C5a. Elastase and cathepsin G may therefore play an important role in the down-regulation of acute inflammation.     

RANTES在银屑病患者角质形成细胞中的表达及其作用

目的：探讨银屑病皮损局部T淋巴细胞高度浸润的原因，并分析了趋化因子RANTES在此过程中的作用。方法：培养银屑病患者皮损处角质形成细胞，通过微孔小室实验检测其上清液对T淋巴细胞的趋化功能；通过酶联免疫吸附(ELISA)法检测上清液中RANTES的表达。结果：银屑病皮损处角质形成细胞培养上清液对T淋巴细胞的趋化能力明显强于正常对照组；其分泌的RANTES水平也高于正常人。结论：银屑病患者皮损局部T淋巴细胞的大量浸润，部分是由于角质形成细胞具有较强的趋化T淋巴细胞能力，而银屑病角质形成细胞中高表达的RANTES可能是发挥此作用趋化因子之一。     

Lack of increase in granulocyte colony-stimulating factor in psoriatic skin.

Previously, we showed an elevated level of pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in psoriatic skin. Granulocyte (G)-CSF, which is also released from the infiltrating cells and epidermal keratinocytes, profoundly influences the biological activities of terminally differentiated neutrophils, in addition to its supporting effects on the proliferation and differentiation of progenitor cells of neutrophil lineage. We have carried out enzyme immunoassay for G-CSF in suction blister fluids and horny tissue extracts from psoriatic skin. Although some samples of the blister fluids and stratum corneum extracts showed G-CSF, there were no significant differences between the concentration in normal and psoriatic skin. These results suggest that, among CSFs, GM-CSF plays a more important role than G-CSF in the local immune responses in psoriasis.     

Angiogenic activity is defective in monocytes from patients with alopecia universalis.

Monocyte/macrophages are important components of cell-mediated immune responses in presentation of antigen, as regulators of lymphocyte function, and as sources of cytokines that modulate functions of cells other than those of the immune system. Their role in the pathogenesis of alopecia areata (AA) and universalis (AU) has not been explored. This study is an investigation of the function of peripheral blood monocytes from normal subjects and patients with AA, AU, and alopecia totalis (AT), with respect to the principal macrophage-derived angiogenic factor, tumor necrosis factor alpha (TNF alpha). Because neovascularization is a necessary component in the anagen phase of hair growth and may play a role in the pathology of these disorders, we asked whether monocyte/macrophage angiogenic activity was compromised in these alopecias. Purified preparations of monocytes were activated in culture. Conditioned media were assessed for angiogenic activity on the chick chorioallantoic membrane and for concentration of TNF alpha by enzyme-linked immunosorbent assay (ELISA). Both angiogenic and the TNF concentration were significantly diminished in conditioned media from AU monocytes when compared to those from normal subjects and patients with AA. These results show that the function of AU monocytes may be abnormal and that the abnormality may distinguish AU from AA. Defective monocyte/macrophage function could also play a pathogenic role via effects on neovascularization and/or modulation of the immune response.     

Enhanced release of inflammatory mediators from lithium-stimulated neutrophils in psoriasis

We have used lithium salts to stimulate degranulation in order to assess neutrophil activity in psoriasis. Evidence is presented for significant enhancement of degranulation of β- glucuronidase (primary granules) and vitamin Bi2-binding protein (secondary granules) from lithium-stimulated neutrophils in psoriatic whole blood. Basal levels of granule markers showed no significant difference between normal and psoriatic neutrophils. On the other hand, enzymes associated with neutrophil function (myeloperoxidase and catalase) were found to be markedly increased in resting psoriatic neutrophils. An important recent development has been the observation that psoriatic neutrophils are ‘activated’ in terms of their neutrophil function. Wahba et al. (1978, 1979) observed enhanced chemotactic and phagocytic properties of isolated psoriatic neutrophils, and Sedgwick, Bergstresser & Hurd (1979, 1980) demonstrated increased adherence of isolated psoriatic neutrophils to glass-wool and nylon fibres. Recently, there have been a number of reports concerning the clearing of psoriasis in patients undergoing haemodialysis and peritoneal dialysis (McEvoy & Kelly, 1976; Buselmeier et al., 1979; Glinski et al., 1979), and it is now believed that improvement was a result of removal of neutrophils rather than some psoriatogenic factor. Lithium salts are commonly used in the treatment of bipolar affective psychosis. Lazarus & Gilgor (1979) drew attention to reports that lithium salts adversely affect the course of psoriasis by direct or indirect mechanisms. They may do so by affecting neutrophil function, thus establishing a relationship between psoriasis and neutrophil activity. Lithium salts can dramatically affect neutrophil activity in terms of granulopoiesis, turnover and migration (Rothstein et al., 1978). We have recently demonstrated that lithium salts have the capacity to induce directly the release of inflammatory mediators from neutrophils in normal whole blood, in the absence of phagocytic stimulation (Bloomfield & Young, 1982). The objective of this investigation was to demonstrate that lithium salts are capable of causing enhanced degranulation from psoriatic neutrophils. In addition, the results would demonstrate in vitro the possible effects which lithium might have in exacerbating psoriasis in vivo, i.e. by causing release of inflammatory mediators from neutrophils which have been shown to be activated.     

银屑病患者角质形成细胞培养上清液对单核细胞形态、表型的影响

目的　探讨银屑病角质形成细胞 (keratinocytes ,KC)在单核细胞向郎格汉斯细胞 (langerhanscells ,LC)分化过程中的作用。方法　从健康志愿者外周血分离单核细胞 ,分别经银屑病患者及正常人KC培养上清单独培养 5天 ,然后通过Leica显微镜观察其形态 ,通过流式细胞仪分析其表型。结果　健康志愿者外周血单核细胞经银屑病患者KC上清培养后 ,细胞的聚集体明显较正常组减少 ,而与阴性对照组间无显著差异 ;CDla、CD80、CD86的表达均较正常人KC上清组减弱。结论　银屑病患者KC中缺乏某些因子而影响了单核细胞向LC的分化 ,或表达了某些因子而抑制了单核细胞向LC的分化。     

Granulocyte-activating mediators (GRAM): III. Further functional characterization of monocyte-derived GRAM

Summary As shown previously monocytes upon stimulation with bacterial lipopolysaccharides (LPS) release granulocyte-activating mediator(s) (M-GRAM) which induced a long-lasting chemiluminescence (CL) response in human granulocytes. M-GRAM could be separated from interleukin-1 and , interleukin-2, interferon and , granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF), since these cytokines were shown to be unable to induce a significant CL response. In contrast, granulocyte macrophage colony stimulating factor (GM-CSF) and particularly tumor necrosis factor (TNF) are important triggers of the oxidative burst and they are capable of inducing a CL response. TNF activity but not lymphotoxin (LT) activity could be demonstrated in M-GRAM samples. A polyclonal rabbit IgG as well as a monoclonal antibody to recombinant human TNF which neutralized the TNF activity in M-GRAM preparations did not substantially block the CL signal. Furthermore, M-GRAM-induced CL response was not significantly inhibited by a polyclonal calf antiserum to human recombinant GM-CSF. For further functional characterization of M-GRAM-induced granulocyte activation different assays were performed in order to compare GM-CSF and TNF: (a) SOD-inhibitable cytochrome C-reduction (^.O₂ ^-); (b) horse radish peroxidase-mediated oxidation of phenol red (H₂O₂); (c) the release of peroxidase; (d) ultrastructural detection of hydrogen peroxide production; and (e) scanning and transmission electron microscopy (SEM and TEM). Significant release of ^.O₂ ^- was induced by M-GRAM, TNF, and GM-CSF, whereas H₂O₂ production was significantly stimulated only by M-GRAM and TNF, as shown by functional and ultrastructural assays. In contrast, only M-GRAM was able to induce significant release of peroxidase. Granulocyte activation could be visualized by SEM and TEM. Upon stimulation with M-GRAM polymorphonuclear neutrophilic granulocytes (PMN) showed an increased adherence to the substratum, developing an increased number of intracytoplasmic vacuoles and short filopodia, whereby the morphological pattern was different from that induced by GM-CSF and TNF. Based on our results we suggest that M-GRAM activity is mediated, in addition to TNF, by a possible new cytokine which is capable to specifically activate granulocytes turning them into scavengers of invading microbes and parasites.The paper was presented in part at the 17th meeting of the ESDR, March 29 – April 1, 1987, Amsterdam, and the 15th meeting of the ADF, November 13–15, 1987, Munich     

Monocyte and neutrophil chemotaxis in psoriasis. Relation to the clinical status and the type of psoriasis

Chemotactic activity of purified monocytes and polymorphonuclear leukocytes was studied in fifty patients with psoriasis vulgaris and in forty-five healthy individuals by an objective in vitro assay with the use of a 51Cr-labeling technic. Both monocytes and polymorphonuclear leukocytes showed a statistically significant increase in chemotactic response, which was positively correlated with disease activity but not with the extent of the cutaneous lesions. The chemotactic activity of monocytes correlated with that of polymorphonuclear leukocytes in the same patients. Exacerbation of psoriasis was preceded by a rapid increase of polymorphonuclear leukocyte chemotaxis, and a decline of chemotaxis occurred during clinical improvement. The psoriatic leukocytes were 22% more sensitive than normal leukocytes to leukotriene B4 than to N-formyl-methionyl-leucyl-phenylalanine. Psoriatic plasma showed chemotaxis-enhancing properties, but only in patients with widespread cutaneous lesions. Additionally, monocyte and polymorphonuclear leukocyte chemotaxis was studied in twenty patients with pustular psoriasis and in fifteen patients with psoriatic arthritis. The chemotactic profiles in pustular psoriasis were different from those in psoriasis vulgaris. Patients with pustular psoriasis had significantly higher polymorphonuclear leukocyte chemotaxis than patients with psoriasis vulgaris, but the chemotactic activity of monocytes was normal. The presence of seronegative arthritis had no influence on chemotactic activity of psoriatic leukocytes.     

Proinflammatory properties of molluscum bodies

Molluscum contagiosum, a condition characterized by benign viral tumours, occasionally becomes inflamed and regresses spontaneously, an event probably initiated by a host cell-mediated immune rejection against the lesion, but it inevitably involves the disruption of the epidermal tissue to expose the molluscum bodies to the tissue fluids of the dermis. It has been suggested that the molluscum bodies induce inflammation by a mechanism similar to that involved in ruptured epidermal cysts or in acne. Despite the occasional development of inflammation in molluscum contagiosum, the proinflammatory properties of molluscum bodies have never been studied in vitro. Thus, in the present study we sought to determine whether molluscum bodies exert a proinflammatory effect by inducing neutrophil chemotaxis. When exposed to fresh serum in vitro, water-insoluble components of molluscum bodies activated the alternative complement pathway to produce chemotactic C5a/C5a des Arg. We also found that an aqueous extract of molluscum bodies exerted potent chemotactic activity for neutrophils. Remarkably high amounts of the immunoreactive proinflammatory cytokines IL-8 and GRO were present in the extract even when compared with psoriatic scale extracts. Gel filtration HPLC of the extract demonstrated the presence of neutrophil chemotactic activity over a wide range of molecular mass. These data suggest that disruption of the epidermal wall of molluscum bodies induces acute inflammatory changes by activation of the alternative complement pathway on exposure to the tissue fluids, and that the molluscum bodies themselves release proinflammatory cytokines and other neutrophil chemotactic factors on decomposition.     

Psoriatic keratinocytes prime neutrophils for an overproduction of superoxide anions

Psoriatic plaques result from an abnormal proliferation of keratinocytes associated with the local presence of T lymphocytes and neutrophils. The exact role of neutrophils in psoriatic lesions remains unclear. The present investigation was aimed at deciphering the capacity of psoriatic keratinocytes to alter in vitro functions of neutrophils. Blood neutrophils from healthy donors were incubated with psoriatic (PK) or healthy keratinocytes (HK) with and without IL-2-activated healthy T lymphocytes. The study was focussed on neutrophil capacity of adherence, viability and superoxide anion production. PK or HK with or without T lymphocytes similarly augmented neutrophil viability after 48 h of co-incubation. PK or HK did not directly activate the superoxide production by neutrophils. However, they both primed neutrophils for an increased fMLF-induced production of superoxide, an effect enhanced by the presence of T lymphocytes. PK were 1.5-fold more efficient than HK to augment this superoxide production. PK cultured with T lymphocytes induced the adhesion of neutrophils 4.7 times more efficiently than HK. The adherence of neutrophils was mediated through ICAM-1, LFA-1 and Mac-1, independently of bioactive lipids. The effects of PK and HK on neutrophil viability and priming were independent of direct cellular contact. In conclusion, keratinocytes can impact neutrophils by increasing their lifespan, and by priming them to overproduce superoxide. PK are more efficient than HK in priming neutrophils, an effect enhanced by T lymphocytes. These results indicate that neutrophils could contribute to psoriasis pathogenesis partly through their pathological interactions with PK.