Properties of murine and human epidermal cell-derived thymocyte-activating factor

Keratinocytes, the predominant cell within the epidermis, perform some macrophage-like functions, such as endocytosis and phagocytosis. We therefore investigated whether keratinocytes may also exert some nonspecific immunoregulatory functions through the secretion of mediators. Tissue cultures of freshly isolated murine and human keratinocytes as well as keratinocyte cell lines secrete a cytokine, epidermal cell-derived thymocyte-activating factor (ETAF), which augments in vitro lymphoproliferative responses. Keratinocyte cell line cells produce increased levels of ETAF activity after exposure to a variety of cell-damaging agents such as silica, endotoxin, phorbol myristate acetate, hydroxyurea, and mechanical disruption. Biochemical studies showed that murine and human ETAF, like interleukin 1 (IL 1), had a molecular weight between 12,000 and 20,000, interacted with hydrophobic phenyl-Sepharose, and was eluted from anion but not cation exchangers. Like IL 1, murine ETAF had a single isoelectric point whereas human ETAF eluted as three peaks of activity (pI 7.2, 5.8, and 5.0). Partially purified ETAF of either species also had the same biological properties of IL 1. That is, ETAF enhanced IL 2 production by T cells in culture, was chemotactic for polymorphonuclear leukocytes, and was directly mitogenic for fibroblasts. When injected into C3H/HeJ mice ETAF induced hepatocyte production of serum amyloid A, an acute phase protein. Furthermore, ETAF, like IL 1, may act as an endogenous pyrogen and induce fever when injected into rabbits. These findings indicate that production of IL 1-like molecules is not confined to cells of the immune system and that ETAF production by keratinocytes may have important implications in would healing, as well in the pathogenesis of inflammatory and neoplastic diseases.     

Human epidermal cells and squamous carcinoma cells synthesize a cytokine that augments natural killer cell activity

Normal as well as transformed epidermal cells (EC) have recently been reported to produce a cytokine--EC-derived thymocyte-activating factor (ETAF), which according to its biologic as well as biochemical properties is indistinguishable from macrophage-derived interleukin 1 (IL 1). In the present study, the effect of supernatants (SN) derived from normal EC and a human squamous carcinoma cell (SCC) line were tested for their effects on natural killer (NK) cell activity. EC- as well as SCC-derived SN were able to augment in vitro NK cell activity of peripheral blood lymphocytes against K 562 cells. In contrast, adherent cell-derived, IL 1-containing SN did not affect NK cell activity. Upon high-pressure liquid chromatography (HPLC) gel filtration, ETAF and the EC-derived NK cell activity-augmenting factor (ENKAF) exhibited a similar m.w. However, by using reverse-phase HPLC, ETAF and ENKAF eluted as distinct peaks of activity, indicating that SCC cell-derived ENKAF is different from ETAF. Furthermore, ENKAF does not contain interleukin 2 (IL 2) or interferon (IFN) activity. The enhancement of NK cell activity was dose dependent and evident after 20 hr of preincubation of effector cells. Pretreatment of target cells with ENKAF did not affect the susceptibility of the target cells. The NK activity of large granular lymphocytes (LGL) purified by discontinuous Percoll gradient centrifugation and further depleted of high-affinity sheep erythrocyte rosetting cells was enhanced by ENKAF. In contrast, no NK cell activity was expressed by LGL-depleted T cell populations before or after treatment with ENKAF. In a single cell cytotoxicity assay in agarose, the number of lymphocyte binding to K 562 was not affected by ENKAF, but the frequency of dead conjugated target cells and presumably of active killer cells was increased by pretreatment with ENKAF. Additional incubation of LGL with ETAF did not further increase ENKAF-mediated augmentation of NK activity. In contrast to ETAF, ENKAF was not chemotactic for polymorphonuclear leukocytes. These results indicate that normal as well as transformed EC release a unique cytokine--ENKAF--which augments NK cell activity of LGL but is distinct from ETAF, IL 2, and IFN.     

Desensitization of animals to the inflammatory effects of ultraviolet radiation is mediated through mechanisms which are distinct from those responsible for endotoxin tolerance

The studies presented in this report indicate that the mechanisms responsible for both ultraviolet radiation (UVR)- and lipopolysaccharide (LPS)-induced desensitization are different from one another and appear to be regulated at the site(s) of administration of the inflammatory agent. Furthermore, desensitization to either UVR or LPS is not due to the inability of interleukin 1 (IL 1)-sensitive target cells within these animals to respond to this endogenous mediator of inflammation. These conclusions were based on the demonstrated ability of UVR-desensitized mice to undergo an acute-phase response after exposure to a systemically administered inflammatory agent (LPS). In a reciprocal manner, LPS-desensitized mice were found to elicit a normal acute-phase response after a single UVR exposure. In addition, both UVR- and LPS-desensitized mice were found to respond normally to the systemic administration of an exogenous source of semi-purified IL 1. Desensitization to the inflammatory properties of either UVR or LPS appears to be controlled at the site of interaction between the tissues capable of producing epidermal-derived thymocyte-activating factor (ETAF)/IL 1 (epidermal keratinocytes or reticuloendothelial cells, respectively) and the exogenous inflammatory stimulus. Peritoneal macrophages obtained from LPS-desensitized mice were found to have a markedly reduced capacity to secrete ETAF/IL 1 in vitro when compared to peritoneal exudate cells (PEC) obtained from normal mice. In parallel with this decreased secretory potential by PEC was the appearance of membrane-associated forms of this mediator. Membrane-associated IL 1 was not found to be present on PEC obtained from normal mice. Keratinocytes obtained from the skin of normal mice or keratinocytes isolated from the irradiated skin site of UVR-desensitized mice were both found to secrete high levels of ETAF/IL 1 constitutively in vitro. Furthermore, both sources of keratinocytes also expressed membrane-associated forms of ETAF/IL 1 constitutively. Therefore, unlike LPS desensitization, the phenomenon of UVR desensitization does not appear to induce changes in the ability of keratinocytes to secrete soluble forms or to express membrane forms of ETAF/IL 1. UVR desensitization may be a result of the inability of ETAF/IL 1 generated within the skin to reach the various IL 1-responsive target cells throughout the body, or may result from the impaired ability of UVR to stimulate ETAF/IL 1 production due to changes in the structure of the skin of chronically UVR-exposed animals.     

Epidermal cells synthesize a cytokine with interleukin 3-like properties

Interleukin 3 (IL 3) is produced by T lymphocytes and T cell lines (EL 4), as well as by a monomyelocytic cell line (WEHI 3), and it activates lymphocytes as well as mast cells. Recently we have demonstrated that epidermal cells (EC) perform monocyte/macrophage-like functions through the release of an interleukin 1-like immunomodulating mediator (EC-derived thymocyte activating factor; ETAF. Because mast cells predominantly are located in the skin, in the present study we investigated whether EC in addition to ETAF may produce IL 3. Normal as well as transformed keratinocytes were able to secrete an IL 3-like mediator (EC IL 3) that induces the proliferation of IL 3-dependent cell lines. Furthermore, both EC IL 3 and WEHI IL 3 have a similar m.w. of 30,000. In addition, an antibody against IL 3 also blocked EC IL 3 activity, suggesting that these molecules appear to be very similar. EC IL 3 production was greatly enhanced by the addition of concanavalin A, phorbol myristate acetate, lipopolysaccharide, and silica. Factor production was completely blocked by inhibiting protein synthesis. These findings demonstrate that keratinocytes synthesize an additional cytokine with the biologic and biochemical properties of IL 3, but distinct from ETAF. Thus, through the production of EC IL 3, EC may participate in the activation of mast cells and thereby mediate inflammatory as well as hypersensitivity reactions.     

Epidermal cell (keratinocyte)-derived thymocyte-activating factor (ETAF)

In order to determine whether keratinocytes play a role in the modulation of the immune response, we investigated the murine keratinocyte cell line Pam 212. In culture these cells generate a substance with a biologic activity that greatly enhances phytohemagglutinin-induced thymocyte proliferation. We have, therefore, called this substance epidermal cell thymocyte-activating factor (ETAF). This keratinocyte-derived supernatant activity is mainly produced at the onset of the logarithmic growth phase and is directly mitogenic for murine thymocytes. Although ETAF by itself exhibits no T cell growth factor activity, ETAF enhances Interleukin 2 production by mitogen-stimulated murine spleen cells. Murine ETAF is not genetically restricted and lacks species specificity since it decreases lectin-induced proliferation of human peripheral blood lymphocytes (as well as murine spleen cells) and also enhances the production of human Interleukin 2. The factor has a m.w. between 15,000 and 25,000 as determined by gel filtration and elutes as a single peak from anion exchange chromatography columns. The activity is maintained mainly at alkaline pH and is rapidly destroyed at temperatures above 60 degrees C. These observations suggest that epidermal cells may interact with the immune system by elaborating nonspecific factors that modulate lymphocyte proliferation and augment lymphokine production.     

Interleukin 1 production by human polymorphonuclear neutrophils

The purpose of this study was to determine whether human polymorphonuclear neutrophils (PMN), which share a common cell lineage with macrophages, could produce factors such as IL 1. Other properties which these two cell types share are their phagocytic nature and the common receptor and antigens on their cell surfaces. IL 1, in many of its physical, biochemical, and functional characteristics, is found to resemble endogenous pyrogen (EP). PMN have been cited as a possible cell source of EP, but there have also been reports in which the capacity of PMN to produce EP has been questioned. This study shows that normal human PMN can be stimulated by particulate agents such as zymosan and soluble agents such as phorbol myristic acetate to produce a factor(s) which induces proliferation of mouse thymocytes, i.e., PMN IL 1. This PMN IL 1 was released from PMN in a dose- and time-dependent fashion. PMN IL 1 was nondialyzable, was heat-labile, and was inactivated at pH below 5 and above 8. PMN IL 1 stimulated the proliferation of normal human synovial fibroblasts and caused release of a neutral protease (plasminogen activator) from synovial cells. The synovial and thymocyte-proliferating capacity of PMN IL 1 was not affected by the protease inhibitor aprotinin or by soybean trypsin inhibitor. Gel filtration studies estimate the m.w. of PMN IL 1 to be approximately 13,000 to 17,000.     

Emigration and accumulation of PMN-leukocytes induced by endotoxin, interleukin 1 and other chemotactic substances

This publication describes polymorphonuclear leukocyte (PMN) emigration and accumulation, which is prerequisite for their defensive function in infected tissues. The extravasated PMNs can kill microorganisms, but in this process they also release proteolytic enzymes and other cell constituents which can alter and even injure the tissues, primarily the microcirculation. In the first part of the paper in vivo quantitation of the acute inflammatory reaction is described with emphasis on PMN emigration and accumulation. With 51Cr-labeled PMNs the kinetics of their emigration induced by a number of chemotaxins and chemotaxinigens was found to be similar, peaking in 1-4 hour old lesions and returning to baseline values thereafter. The most potent substance tested was endotoxin, which induced a PMN influx at a molar concentration a least 3 orders of magnitude lower than the other substances tested, implying the these substances are not the primary endogenous mediators of endotoxin induced inflammation. Next we describe an observation which shed considerable light on the mechanisms underlying PMN emigration. When a chemotaxin or endotoxin was injected intradermally and after varying periods of time reinjected into the same site, the PMN influx into those sites was diminished, compared to sites not previously injected, i. e. injected for the first time. This tachyphylaxis or diminished responsiveness was attributed to a downregulation of receptors, presumably on endothelial cells, coupled to a facilitatory mechanism. Other mechanism proposed to terminate emigration of PMNs during inflammatory reaction were unlikely, based on our experimental findings. Endotoxin is not chemotactic in vitro but it induces PMN emigration when injected intradermally. Hence the third part of the publication deals with PMN emigration induced by interleukin 1 and its significance for endotoxin-induced inflammation. IL 1 is the only chemotaxin which induces PMN accumulation at a concentration comparable to that of endotoxin and considerably lower than the other chemotaxins. There was cross tachyphylaxis between endotoxin and IL 1 and vice versa. The PMN influx into IL 1 sites injected 6 hours earlier with IL 1 or with endotoxin was diminished compared to IL 1 sites injected into normal skin. Sites injected first with IL 1 and then with a low dose of endotoxin also exhibited cross tachyphylaxis. FMLP or LTB4 injected into sites pretreated with endotoxin did not exhibit cross tachyphylaxis, i. e. the PMN influx was similar to sites injected for the first time with these chemotaxins.(ABSTRACT TRUNCATED AT 400 WORDS)     

The TNF Receptors p55 and p75 Mediate Chemotaxis of PMN Induced by TNFalpha and a TNFalpha 36-62 Peptide

The present study was performed to examine whether residues 36-62 of TNFalpha contain the chemotactic domain of TNFalpha, and whether the p55 and p75 TNF receptors are involved in TNFalpha induced chemotaxis. The chemotactic effect of TNFalpha on PMN was inhibited by the mAbs Hrt-7b and Utr-1, against the p55 and p75 TNF receptors, respectively. Both receptors may therefore be required for mediating the chemotactic effect of TNFcz. The synthetic TNFalpha 36-62, similar to TNFalpha, had chemotactic effects on both PMN and monocytes. The chemotactic activity of the TNFalpha 36-62 peptide on PMN, was inhibited by Htr-7b, Utr-1 and soluble p55 receptor, which shows that the peptide possessed the ability to induce chemotaxis through the TNF receptors. In contrast to TNFalpha, the peptide did not show a cytotoxic activity against WEHI 164 flbrosarcoma cells. It is suggested that different domains of the TNFalpha molecule induce distinct biological effects.     

Generation of biologically active, complement-(C5) derived peptides by cathepsin H

The thiol proteinase cathepsin H, isolated and purified from rat liver lysosomes, provokes acute inflammation characterized by the accumulation of polymorphonuclear leukocytes (PMN) when injected intracutaneously into newborn rats. We have examined the possibility that the accumulation of PMN at skin sites injected with cathepsin H is due, in part, to generation locally of C-derived chemotactic factors. We have found that cathepsin H acts in a concentration- and time-dependent fashion in whole human (and rat) EDTA-plasma to generate C5-derived peptides with chemotactic activity for PMN. Chemotactic activity was not generated in EDTA-plasma by either heat-inactivated cathepsin H or by a combination of active enzyme and a thiol proteinase inhibitor isolated from rat epidermis. Cathepsin H also acted in a concentration- and time-dependent fashion on isolated (functionally pure) human C5 to yield chemotactic activity for PMN as well as PMN lysosomal enzyme-releasing activity. Whereas 10 ng/ml cathepsin H generated significant chemotactic activity from isolated C5 (1000 CH50 U/ml), 7 to 10 micrograms/ml were required to generate chemotactic activity in whole EDTA-plasma. Cathepsin H not only was capable of generating biologically active, C5-derived peptides, but also was capable of degrading these peptides. Incubation of either whole EDTA-plasma or isolated C5 with high concentrations of cathepsin H (e.g., 25 micrograms/ml and 100 ng/ml, respectively) caused the rapid appearance of chemotactic activity followed by an equally rapid disappearance. PMN accumulated more rapidly in the skin of newborn rats injected with cathepsin H-treated C5 than in the skin of animals injected with cathepsin H alone. These data suggest that generation by cathepsin H of C-derived chemotactic activity contributes to the ability of this enzyme to induce dermal inflammation.     

Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin 8

Human articular chondrocytes, when stimulated with interleukin 1 beta (IL 1 beta), tumor necrosis factor-alpha (TNF-alpha), or with the double stranded RNA poly (rI).poly (rC), produce a chemotactic activity for granulocytes. The induction with IL 1 beta could be abolished by an antibody to IL 1 beta but not by an antibody to interleukin 6 (IL 6), indicating that the latter is not a mediator for the production of chemotactic activity. The inducers had no direct chemotactic effect on granulocytes. The granulocyte chemotactic factor from chondrocytes was characterized with a specific antibody against leukocyte-derived interleukin 8 (IL 8). The specificity of this antibody was demonstrated by immunochemical and biological criteria such that it could immunoprecipitate only the 6-7 kDa IL 8 protein from fibroblasts, and that it did not neutralize a structurally related monocyte chemotactic protein. This antibody against IL 8 completely neutralized the granulocyte chemotactic activity from stimulated chondrocytes. This demonstrates the identity of chondrocyte IL 8 with leukocyte- and fibroblast-derived IL 8. Our data show that leukocyte chemotaxis into the inflamed joint can be mediated by IL 8, induced in both synovial fibroblasts and chondrocytes by the inflammatory cytokines IL 1 and TNF-alpha.     

A new role for epidermal cell-derived thymocyte activating factor/IL-1 as an antagonist for distinct epidermal cell function

It is known that many immunologic responses to IL-1 are antagonized by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH). This led us to investigate the possible reciprocal effects of IL-1 and the functionally related epidermal cytokines, epidermal cell-derived thymocyte activating factor (ETAF) and IL-6, on the melanogenic effect of alpha-MSH on murine Cloudman melanoma cells. When these cells were treated with ETAF in combination with alpha-MSH or its potent analog [Nle4,D-Phe7]-alpha-MSH, the melanotropin induced increase in tyrosinase activity, and thus melanin synthesis, was abrogated. This inhibitory effect of ETAF was not mediated by competitive binding to the melanotropin receptor, because ETAF also blocked the melanogenic response of melanoma cells to isobutyl methylxanthine (IBMX) and to PGE1 and PGE2. ETAF had no effect on cellular proliferation. Inhibition of the stimulated tyrosinase activity by ETAF was not due to diminished cAMP synthesis or increased cAMP degradation. Cells treated concomitantly with ETAF and alpha-MSH, IBMX, or PGE1 had the same cAMP levels as cells treated with alpha-MSH, IBMX, or PGE1 alone. In contrast to ETAF, human rIL-1 alpha or IL-1 beta alone or in combination did not have an inhibitory effect on melanogenesis. IL-6 significantly inhibited the basal level of tyrosinase and partially abrogated the alpha-MSH-induced tyrosinase activity. IL-6 also stimulated cellular proliferation when added alone or in combination with alpha-MSH. Granulocyte-macrophage colony stimulating factor (GM-CSF) did not alter either the tyrosinase activity or cellular replication at the concentrations tested. IL-1 alpha, GM-CSF, and IL-6 or IL-1 alpha and GM-CSF added together did not significantly affect the MSH-induced tyrosinase activity. These results ascribe a new potential function for ETAF and IL-6 as modulators of the melanogenic response of pigment cells.     

Suppressive effect of ultraviolet-B-irradiation of epidermal cells on the induction of contact sensitivity

Contact sensitivity to trinitrophenyl (TNP) hapten was induced by subcutaneous (s.c.) administration of TNP-modified syngeneic spleen cells or epidermal cells (EC) (TNP-EC). Intraperitoneal (i.p.) inoculation of TNP-EC resulted in a comparable response, whereas i.p. administration of TNP-spleen cells or TNP-modified-ultraviolet (UV)-preirradiated EC (TNP-UV-EC) failed to induce TNP-contact sensitivity responses. The present study investigates the effect of UV-irradiation on the potential of EC for inducing the contact sensitivity response. Exposure of BALB/c mouse EC in vitro to 1600 J/m2 of UV-B before they were modified with TNP had no discernible effect on the Ia-positivity and viability of EC. Coexistence of TNP-UV-EC had no inhibitory effect upon the contact sensitivity response induced by TNP-EC via the i.p. route. The absence of suppressor cell generation was substantiated by the adoptive transfer of spleen cells from mice administered TNP-UV-EC i.p. to normal syngeneic mice. The effect of interleukin 1 (IL-1) or epidermal cell-derived thymocyte-activating factor (ETAF) in restoring the ability of TNP-UV-EC to induce contact sensitivity was examined. IL-1 or ETAF administered along with TNP-spleen cells i.p. induced a potent contact sensitivity response, whereas the same preparations of IL-1 or ETAF were unable to restore the contact sensitivity induction by TNP-UV-EC. The results are discussed in the context of UV-induced cell surface changes of the Langerhans cell population.     

Recombinant human tumor necrosis factor alpha lacks chemotactic activity for human peripheral blood neutrophils and monocytes

Preparations of recombinant human tumor necrosis factor alpha (rhuTNF alpha) free of aminoterminal methionine were tested for human neutrophil granulocyte (PMN) and monocyte (MO) chemotactic activity using the Boyden chamber system. Over a wide range of concentrations (10(-7)-10(-15) M) rhuTNF alpha of two different sources failed to elicit chemotactic responses in PMN or MO, whereas strong PMN and MO chemotactic activity could be detected using the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In addition, rhuTNF alpha containing 62% aminoterminal methionine failed to induce PMN and MO chemotaxis. It is concluded that rhuTNF alpha may not be a chemotaxin for human PMN and MO in vitro.     

Normal human neutrophils are a source of a specific interleukin 1 inhibitor

In the course of our study on neutrophil production of an interleukin 1 (IL-1)-like factor, we found that the addition of polymorphonuclear neutrophils (PMN) to monocytes cultured in the presence of zymosan resulted in decreased IL 1 activity of the resultant supernatant, suggesting that PMN may contain an inhibitor of IL 1. The objective of this investigation was to study this IL 1 inhibitor which normal human PMN contain. The inhibitor is constitutively present in the PMN because 0 hr PMN lysates and unstimulated PMN supernatants also show inhibitory activity. The PMN inhibitor inhibits IL 1 (crude and partially purified) in a dose-response manner and does not affect basal [3H]thymidine incorporation in the presence or absence of PHA-P. The PMN inhibitor does not have any effect on interleukin 2 (IL 2)-induced proliferation of the IL 2-dependent CTLL cells. The inhibitor can be generated in the absence of serum and is not produced as a result of proteolytic activity from PMN enzymes. The inhibitor is heat-labile and is most stable at neutral pH. Gel filtration studies on Sephadex G-200 indicate that the inhibitor is heterogeneous in size. Two inhibitory peaks, at 45,000 to 70,000 m.w. and at greater than 160,000 m.w., were observed. When zymosan-stimulated PMN supernatant was chromatographed, there was separation of inhibitory factor from a 17,000 m.w. proliferating factor. Presence of this PMN inhibitor may be important in negative regulation of IL 1.     

Human hepatocytes metabolizing ethanol generate a non-polar chemotactic factor for human neutrophils

When human hepatocytes were incubated with low concentrations of ethanol they general chemotactic activity for human neutrophils. Generation of chemotactic activity was dependent upon duration of incubation and concentration of ethanol used. Production of chemotactic activity by ethanol-treated hepatocytes was inhibited completely in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole. PMN isolated from rats, in contrast, do not respond chemotactically to the factor released by homologous cells. Preliminary studies indicated that the chemotactic factor is non-polar in nature (perhaps related to leukotriene B4). These results indicate that human hepatocytes, when exposed to ethanol, generate chemotactic factor(s) for human PMN. The occurrence of this phenomenon may explain, in part, the PMN infiltrates observed in human liver during the course of acute alcoholic hepatitis.     

Polymorphonuclear leukocytes modulate tissue factor production by mononuclear cells: role of reactive oxygen species

To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures, PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio. When the ratio increased from 1/1000 to 1/5, without or with LPS, the TF activity of PBMC increased to peak at 2.5-fold the baseline value (p < 0.01). The TF Ag and TF mRNA also increased. This potentiating effect was mediated by reactive oxygen species (ROS) released by PMN during the coculture; it did not require direct cell contact between PMN and PBMC, it was enhanced when PMN were stimulated by fMLP (a chemotactic peptide), and it was inhibited by two antioxidants, N-acetyl cysteine and pyrrolidine dithiocarbamate. In contrast, when the PMN/PBMC ratio was further increased from 1/2 to 2/1, the PBMC TF activity, Ag, and mRNA decreased and were inhibited compared with those of PBMC cultured alone (p < 0.01). This inhibitory effect required direct cell contact between PMN and PBMC, and it was not due to a PMN-mediated cytotoxicity. To confirm the role of ROS, H2O2 enhanced then inhibited the TF activity of PBMC in a dose-dependent manner, similarly to PMN. Thus, PMN may play an important role in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production by PBMC via the release of ROS.     

Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1)

LPS stimulated human blood mononuclear leukocytes to produce a chemotactic factor for human neutrophils. The effect of LPS was dose-dependent; 10 micrograms/ml was optimal for production of chemotactic factor. Chemotactic activity was detected 3 hr after LPS stimulation, and reached its peak at 12 hr. No activity was detected in culture supernatants of unstimulated cells, provided LPS-free media were selected. Isoelectric point of the factor, determined by chromatofocusing, was approximately 8 to 8.5. Molecular weight was approximately 10 kilodaltons by Sephacryl S-200 gel filtration or by HPLC gel filtration on TSK-2000 and -3000 columns in succession. The gel filtration fractions were also assayed for IL 1 activity. The elution position of IL 1 activity corresponded to a m.w. of 18. There was no chemotactic activity in the IL 1 activity peak. Furthermore, highly purified natural Il 1 alpha and -beta and recombinant Il 1 alpha and -beta did not exhibit chemotactic activity for neutrophils in our assay. Among mononuclear leukocytes, the monocyte was the principal producer of neutrophil chemotactic factor. These results suggest that a chemotactic factor for neutrophils, different from IL 1, is produced by LPS-stimulated blood monocytes.     

Neutrophil-activating peptide 1/interleukin 8 mRNA expression and protein secretion by human monocytes: effect of cyclosporin A

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) is a recently described cytokine with potent chemotactic activity for human neutrophil granulocytes (PMN) and T cells. In psoriasis, a chronic hyperproliferative and inflammatory skin disorder, PMN and T cells are found as prominent cells in the inflammatory infiltrate of the lesions; however, monocytes were shown to be the first cells invading a newly formed plaque. NAP-1/IL-8 was found to be present in high amounts in the skin and in scale material of psoriatic patients. Psoriasis responds well to systemic treatment with cyclosporin A (CsA), an immunosuppressive peptide. Therefore, we addressed the question of whether the clinical improvement of psoriatic patients during CsA therapy may be due to an inhibition of NAP-1/IL-8 production and secretion from monocytes. Purified human monocytes were stimulated by lipopolysaccharide in the presence or absence of various concentrations of CsA. Production of NAP-1/IL-8 was determined as expression of specific mRNA by fluorescent in situ hybridization. Secreted peptide was measured by bioassay (PMN chemotaxis) and enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies. The results show that CsA neither inhibited mRNA expression for NAP-1/IL-8 nor secretion of the peptide. These findings support the hypothesis that the pharmacological effect of CsA may be restricted to the inhibition of T-cell activation and proliferation.     

Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.