Isolation and characterization of permeability factors from rabbit neutrophils.

Four basic proteins that increase vascular permeability have been isolated in purified form from rabbit neutrophilic granules. These proteins are termed band 1, 2, 3, and 4 protein according to their electrophoretic migration in acrylamide gel. Molecular weights of band 1 and 2 protein derived from amino acid composition were 4800 and 5300, respectively. These values are in good agreement with those obtained for these proteins by gel diffusion techniques. The molecular weight of band 3 protein was also in the range of 5000 by the latter technique. The molecular weight of band 4 protein determined by ultracentrifugal analysis and amino acid composition was 12,000. Although all four proteins had the capacity to induce immediate increase in vascular permeability, only band 2 protein was found to release histamine from isolated rat peritoneal mast cells. Furthermore, it has been shown that the permeability-inducing activity of band 2 protein can be inhibited by pretreating rabbits with antihistamine. Band 2 protein did not release histamine from rabbit platelets and depletion of rabbit platelets from the circulation had no influence on the permeability-inducing activity of this protein. Band 1, 3, and 4 proteins did not release histamine from isolated rat peritoneal mast cells and their capacity to increase vascular permeability remained unaffected by treatment of rabbits with antihistamine. These investigations suggest that the histamine-releasing activity of band 2 protein is a specific phenomenon and is associated with particular amino acid grouping or spacial configuration of the molecules. By the same token, the increase in vascular permeability induced by the nonhistamine-releasing band 1, 3, and 4 proteins represents a specific phenomenon (or phenomena) not particularly related to the over-all charge of these molecules.

Effects of ultraviolet-visible irradiation in the presence of melanin isolated from human black or red hair upon Ehrlich ascites carcinoma cells.

The present study is an attempt to investigate the possibility that ultraviolet irradiation in the presence of pheomelanin may be more harmful to cells than the irradiation in the presence of eumelanin. The effects of UV-visible irradiation upon Ehrlich ascites carcinoma cells in the presence of the melanin isolated from human black hair (eumelanin) or from red hair (pheomelanin) were investigated. Irradiation of these cells was found to produce cell lysis, as observed by leakage of 51Cr from labeled cells and intracellular lactic dehydrogenase from the cells and decrease in cell viability demonstrated by the trypan blue exclusion test. The three parameters were quantitatively parallel to one another under various experimental conditions, namely different periods of irradiation and irradiation in the presence of different concentrations of melanin. The above effects were more pronounced when the irradiation was carried out in the presence of melanin from red hair than in the presence of black-hair melanin. In the absence of either melanin, the irradiation did not produce any significant effect in cell viability or cell lysis. Irradiation of the cells in the presence of red-hair melanin also decreased the transplantability of these cells. These observations clearly show that irradiation of cells in the presence of pheomelanin could produce cytotoxic effects. The present experimental design may have application in the development of in vitro models for the study of UV radiation-induced cutaneous carcinogenesis. The reactions of pheomelanin may be related to the susceptibility of "Celtic" skin to UV radiation-induced skin damage and carcinogenesis.

Effects of melanin-induced free radicals on the isolated rat peritoneal mast cells.

Pheomelanin from human red hair (RHM) produces considerably more cellular damage in Ehrlich ascites carcinoma cells when subjected to radiations of wavelength 320-700 nm than eumelanin from black hair (BHM). Irradiation of RHM generated large amounts of superoxide while BHM did not produce detectable amounts of superoxide. The present investigations describe the effects of irradiation of mast cells in the presence of various natural and synthetic melanins. Irradiation of mast cells in the presence of RHM and red hair melanoprotein released large amounts of histamine while BHM and synthetic melanins prepared from dopa, cysteinyldopa, or a mixture of dopa and cysteinyldopa did not release histamine. The release of histamine at lower concentrations of RHM was not accompanied by the release of 51Cr from chromium-loaded cells, suggesting that this release was of noncytotoxic nature. On the other hand, the release of histamine at higher concentrations of RHM was due to cell lysis since both histamine and cytoplasmic marker 51Cr were released to the same extent. The release evoked by large concentration RHM was not inhibited by superoxide dismutase or catalase. This suggests that the cell lysis under these conditions was not due to H2O2 or O-2. The finding that mast cells release histamine when irradiated in the presence of RHM suggests that the immediate and late-phase reactions seen in sunburn may in part be due to the release of mediators from these cells.

Differential effects of antioxidants and indomethacin on compound 48/80 induced histamine release and Ca2+ uptake in rat mast cells.

The effect of nordihydroguaiaretic acid (NDGA), vitamin E, butylated hydroxytoluene (BHT) and indomethacin on histamine release and Ca2+ uptake in rat mast cells stimulated with compound 48/80 was studied. NDGA inhibited both the release of histamine and Ca2+ uptake in stimulated cells; however, there was no correlation between inhibition of Ca2+ uptake and the amount of histamine release. At a concentration of 5 microM, NDGA completely inhibited Ca2+ uptake, while histamine release was decreased by less than 50%. BHT (50 microM) inhibited both the Ca2+ uptake and histamine release. On the other hand, vitamin E (50 microM) inhibited histamine release by 70% without impairment in Ca2+ uptake. In the absence of the stimulus, vitamin E increased the cell-associated Ca2+; however, it had no effect on spontaneous release of histamine. Indomethacin (3 microM) inhibited Ca2+ uptake in stimulated cells by 50%, but did not affect the release of histamine. The results suggest that a part of Ca2+-influx may not be related to the coupled activation--secretion response and that lipid peroxidation through the lipoxygenase pathway may be involved in secretion of histamine from mast cells.

Modulation of 8-methoxypsoralen-photoinduced cutaneous inflammatory reactions by various chemotherapeutic agents in vivo.

Exposure of albino rabbits to UVA-VIS (320-700 nm) radiation after the topical application of 8-methoxypsoralen (8-MOP) cream is associated with acute cutaneous inflammatory reactions in situ. In the present studies the effects of various agents on 8-MOP plus light induced cutaneous inflammatory response viz. increase in vascular permeability (iVP), accumulation of polymorphonuclear leukocytes (aPMN) and erythema formation were investigated. The inflammatory reactions were induced by a single exposure of 8-MOP-sensitized sites to UVA-VIS (9.4J/cm2) light. Indomethacin, p-bromophenacyl bromide (BPAB), MK886 (trade name of Merck Sharpe & Dome), ibuprofen (IB), nordihydroguaiaretic acid (NDGA) or quinacrine were applied topically in cream base at various times prior to 8-MOP application. The iVP and aPMN were quantitated 24 h postirradiation using 125I-HSA and 51Cr-labeled PMN respectively, while erythema was graded visually. The rate of iVP, aPMN and erythema was inhibited almost completely by indomethacin (7.5-10%) when applied twice, 18 h and 3 h prior to 8-MOP. At lower concentrations of indomethacin (< or = 5%) iVP was inhibited whereas aPMN was augmented. The BPAB (0.25%) inhibited more than 90% of 8-MOP-photoinduced iVP and aPMN while there was partial reduction in erythema. The MK886 (0.1%) cream inhibited about 50% of iVP and aPMN but erythema persisted. The agents that are somewhat nonspecific such as IB, quinacrine and NDGA inhibited 8-MOP-photoinduced inflammation only marginally at the concentrations tested. The fact that iVP, aPMN and erythema can be dissociated suggests that there are independent variables in 8-MOP-photoinduced reactions, which involve multifactorial mechanisms probably controlled by different cell-signalling pathways and mediators.

Modulation of phagocytosis and intracellular bactericidal activity of polymorphonuclear and mononuclear cells by cationic proteins from human granulocytes: alternative pathway of phagocytic enhancement.

Cationic lysosomal proteins from human polymorphonuclears (PMN) were isolated by column chromatography and divided into five fractions. On acrylamide gel electrophoresis, fraction I had four bands slower than lysozyme (LZM) mobility; fraction II had five or six bands slower than LZM; fraction III had at least seven bands slower and two bands faster than LZM; fraction IV contained LZM, two bands faster and a few faint bands slower than LZM; fraction V was composed of almost pure LZM. Partial characterization of the fractions showed presence of neutral protease in fractions I-IV, chymotrypsin in fraction III, lysozyme in fractions IV and V, and phospholipase A2 mainly in fractions II and III. Modulatory activity of fractions I-V were tested at concentrations up to 50 micrograms/ml. Enhancement of phagocytosis of Staphylococcus aureus was observed by fractions I, IV, and V, whereas phagocytic index was enhanced by all but the fraction II. Intracellular bactericidal activity (ICBA) was markedly enhanced by fractions I, II, and V. Addition of DNA or cytochalasine B inhibited or abolished phagocytosis-enhancing activity of cationic fractions. Their influence on ICBA was much less pronounced. Fraction III enhanced phagocytic index and phagocytosis of E. coli, whereas fractions I and II enhanced intracellular bactericidal activity against this bacteria. Enhancement of phagocytic activity of monocytes has also been observed. The data suggest that some cationic lysosomal fractions from human PMNs enhance phagocytosis and phagocytic index by human PMNs and monocytes and intracellular bactericidal activity of human PMNs. This alternative pathway of phagocytic enhancement is unrelated to the previously described enhancers of phagocytosis and may play a role in defense mechanisms against infection.

Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis.

The cationic proteins from neutrophil lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O2- by human PMNs during phagocytosis. Human PMNs were reacted with different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O2- generated was determined by superoxide dismutase inhibitable reduction of cytochrome c. Total cationic protein extract from neutrophil lysosomes enhanced O2- generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O2- generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochrome c reduction by O2- formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O2- generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O2(-)-enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O2- generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O2- generation. Electrophoretic analysis of the isolated protein fractions suggests that O2- generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.

Influence of cationic superoxide generation enhancing protein (SGEP) on phagocytic and intracellular bactericidal activity of human polymorphonuclear cells.

Cationic fraction III from the lysosomes of normal human peripheral blood polymorphonuclear cells (PMNs) was found to contain superoxide generation enhancing protein (SGEP). Herein, we report on the influence of partially purified SGEP obtained from fraction III (subfractions III-5 and III-6), on various phagocytic functions of human PMNs. SGEP markedly enhanced intracellular bactericidal activity of human peripheral PMNs. The enhancement was time and dose dependent. It also reduced adhesiveness of the PMNs. SGEP did not influence chemotaxis, phagocytosis or phagocytic index. These findings are compatible with our original observation regarding superoxide generation enhancement properties of SGEP.

Desensitization of rabbit skin by repeated exposure to UV-visible light of sites injected with Rose Bengal.

We have shown in a previous paper that irradiation of rabbit skin sites injected with Rose Bengal (RB) produces immediate increase in vascular permeability and accumulation of PMNs. Studies on the development of temporary tolerance and the biological parameters related to the development of such tolerant state by repeated exposure to light of RB-injected sites are reported here. The increase in VP and PMN migration induced by RB (10 nmol) are of an immediate nature, i.e., occur within the first 3 h of irradiation, and the reaction subsides gradually after 24 h. When such moderate insult is repeated, the skin becomes tolerant to subsequent exposure to light in the presence of RB. This tolerant state is temporary, i.e., the desensitized sites are fully recovered in 72 h. The loss of responsiveness of RB-injected sites previously exposed to light was not due to diffusion of the injected dye from the sites since reinjected sites also showed reduced response and the sites injected three days before but not irradiated showed normal response. The sites that were made tolerant to RB-induced phototoxic reactions, when injected with compound 48/80, an agent known to degranulate mast cells, did not show an increase in VP. This suggests that either the mast cells were depleted from the sites or the mast cells in the sites were rendered refractory by previous exposure to light. It was also found that the sites made tolerant to RB plus light were unresponsive to exogenously injected histamine. The sites tolerant to RB plus light when injected with zymosan-activated serum (ZAS) did not stimulate the migration of PMNs. This loss of chemotactic response to ZAS may have relevance to photodamage of vascular endothelium. These observations are discussed in relation to the development of the tolerant state by repeated exposures to subthreshold doses of light in solar urticaria.

Quantitation of cutaneous inflammation induced by reactive species generated by UV-visible irradiation of rose bengal.

The present studies were undertaken to quantitate the initial inflammatory response produced by the photo-generated reactive species in rabbit skin. Rose bengal (RB), a photosensitizer dye, was injected into the skin sites at various concentrations and exposed to UV-visible light for 30-120 min. The increase in vascular permeability and the accumulation of PMNs were investigated using 125I-labeled albumin and 51Cr-labeled PMNs. RB at a concentration of 1 nmol with 120-min exposure to light enhanced vascular permeability by 3.7 times and accumulation of PMNs by 3.3 times. As low as 0.01 nmol of RB produced discernible effects. beta-Carotene (0.1 nmole) inhibited the inflammatory response by 75-100%, suggesting that the reactive species involved in this response was predominantly singlet oxygen. The increase in vascular permeability was inhibited by 48-70% by 25 micrograms of chlorpheniramine maleate. It is therefore suggested that histamine plays a major role in the initial vascular response. The studies demonstrate that this rabbit model is suitable for the quantitation of photoinduced inflammatory response which is not observable by gross anatomic procedures.

Comparative studies on the tolerance to photoinduced cutaneous inflammatory reactions by psoralen and rose bengal.

The photochemotherapeutic value of topical 8-methoxypsoralen (8-MOP) plus UVA irradiation has been well recognized. The phototoxicity associated with psoralen plus UVA (PUVA) therapy is hallmarked by an increase in vascular permeability (iVP), the accumulation of polymorphonuclear leukocytes (aPMN) and erythema formation in situ. Rose bengal (RB) plus UVA-VIS light (320-700 nm) produces a similar acute inflammatory response, but without immediate or delayed erythema and perceptible edema. This study describes some of the parameters involved in inflammatory reactions evoked by PUVA and the results are compared with RB-induced phototoxic reactions. The rates of iVP and aPMN with a 3 h pulse were quantified using 125I-albumin and 51Cr-labelled PMNs respectively. The erythemal response was graded visually. 8-MOP cream was applied topically, while RB was injected intradermally in rabbit skin before UVA-VIS (9.4 J cm-2) irradiation. The data show that there is no significant difference in the rates of iVP, aPMN and erythema formation between normal skin sites and mast cell-depleted skin sites when challenged with 8-MOP plus light. These results suggest that in situ mast cells do not play a significant role in 8-MOP-photoinduced acute cutaneous inflammatory reactions, in contrast with RB-photoinduced reactions. The iVP and aPMN responses are minimal or absent in sites subjected to repeated exposure to 8-MOP plus light for three or more consecutive days, suggesting the establishment of a desensitized/unresponsive state. Moreover, 8-MOP-photo-desensitized sites do not produce iVP and aPMN of the same magnitude as the normal (naive) skin sites when challenged with RB plus light. Similarly, RB-photo-desensitized sites do not produce iVP and aPMN of the same magnitude as the native skin sites when challenged with 8-MOP plus light. The desensitization and cross-desensitization of skin sites to 8-MOP- or RB-photoinduced reactions suggest that there is either direct attack on the target cell(s), thereby removing the ability to express adhesion molecules, such as endothelial leukocyte adhesion molecule 1 (ELAM-1) or intercellular adhesion molecule 1 (ICAM-1), involved in the accumulation of inflammatory cells, or downregulation of the secretion/release of putative agent(s), such as interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), responsible for the initiation and progression of cutaneous inflammations.

Photoinduced cutaneous inflammatory response by psoralens.

Our studies describe the inflammatory response in rabbit skin induced by topical application of 8-methoxypsoralen (8-MOP) and UVA-visible irradiation (320-700 nm). Increase in vascular permeability (iVP) and accumulation of polymorphonuclear leucocytes (aPMN) at the test sites were quantitated using 125I-albumin and 51Cr-labelled PMNs respectively. Erythema was graded visually. 8-MOP cream was applied topically and irradiated. The erythemal response, aPMN and iVP at the test sites were quantitated at 6, 24, 48 and 72 h post-irradiation. The iVP and aPMN were maximal at 24 h; the erythemal response was the same at 24-48 h. The responses were dependent on 8-MOP concentration and irradiation dose. Topical application of 200 micrograms 8-MOP cream followed by irradiation for 2 h (9.4 J cm-2) produced 3-7 times iVP, 2-4 times aPMN and intense erythema at the test sites after 24 h. Neither aPMN nor iVP was detected before 6 h and erythemal response was not observable up to 16 h after irradiation. The aPMN and iVP gradually subsided in 72 h, although the erythemal response was still present. The repeated exposure of 8-MOP-treated sites for three consecutive days 24 h apart did not produce appreciable iVP or aPMN at 72 h or 24 h after the last exposure; however, erythema persisted. The 8-MOP-treated sites previously exposed for three consecutive days on reapplication of 8-MOP cream plus irradiation showed significantly less response compared with non-pretreated sites. Our results suggest that the erythemal response is not directly related to either iVP or aPMN.

Histamine levels in middle ear effusions.

The presence of histamine in 131 middle ear effusions was determined by the fluorometric assay technique. This potent mediator of inflammation was found in significant amounts in most of the samples, suggesting that it may play an important role in the pathogenesis of otitis media with effusion. It is postulated that mast cells located in the lamina propria of the tympanic mucoperiosteum are triggered to degranulate and release histamine by anaphylatoxin derived from activation of the complement system.

Role of lysosomal constituents of neutrophils in corneal graft reaction.

Band 2 protein exists in a firmly bound state in lysosomes, and is released when the cells concerned are specifically stimulated. The necessary stimulus can originate from different kinds of immunogenic interactions. The objective of this study was to determine whether during corneal graft reaction Band 2 protein could be detected in the host's cornea and aqueous humour. Band 2 protein was detected in the reacting host corneas (8 out of 23 eyes) and the aqueous humours (2 out of 23 eyes), mainly during the second and third postoperative weeks when parallel histological studies showed an accumulation of neutrophils in the graft bed. The severity of graft reaction appeared to run parallel to the amount of free Band 2 protein in the host cornea. After this period, in spite of the presence of a large number of neutrophils in the reacting host cornea, Band 2 protein was hardly detectable. This study indicates that the necessary stimulus for the secretion of Band 2 protein from rabbit neutrophils is present in the early stages of corneal xenograft reaction. The presence of the protein in the host cornea may be a factor in the development of the vascular response associated with the corneal graft reaction.

Phagocytosis by retinal pigment epithelium: evaluation of modulating agents with an organ culture model.

Several drugs known to modulate the phagocytic activity of "professional" phagocytes were examined for effects on the bovine retinal pigment epithelium (RPE). The test system employed was an organ culture model (the RPE cup) developed previously. Iodoacetate depressed the ability of the RPE to phagocytose latex particles labelled with iodine 125, as measured by the gamma-emissions from the RPE cup. Indomethacin, sodium salicylate, azathioprine and alcohol also showed depressant effects. Hydrocortisone sodium succinate had no effect, as has previously been reported. Bestatin, an immunostimulator, did not have a statistically significant effect. The results establish the phagocytic role of the RPE and suggest that this activity may be chemically modulated. This inference may have potential application in the study of retinal dystrophy.

Damage to corneal endothelial cells by lysosomal enzymes in stored human eyes.

We wished to see if the concentration of lysosomal enzymes in the aqueous humour had any relation to the number of dead corneal endothelial cells in stored eyes from human donors. Forty pairs of eyes were obtained: one eye of each pair was tested immediately and the other was tested after storage in a moist chamber at 4 degrees C for up to 10 days. The aqueous humour of each eye was aspirated and the concentration of acid phosphatase (a marker of lysosomal hydrolytic enzymes) was measured. Simultaneously the numbers of dead and living cells per unit area of the corneal endothelium were counted following their staining with trypan blue and p-nitroblue tetrazolium respectively. As the storage time increased, the concentration of acid phosphatase in the aqueous humour increased and the number of living corneal endothelial cells decreased. The number of living cells decreased to about 50% at an average enzyme concentration of 9 X 10(-3) muM/h. The eyes stored for less than 3 days were the least damaged.

The prevention of autolysis of stored cornea using steroid as a lysosome membrane stabilizer.

Many eyes donated for use in corneal grafting are rejected because of signs of autolysis in the donor material. The purpose of this experimental study was to determine whether hydrocortisone acting as a lysosome membrane stabilizer could prevent or retard autolysis of the corneas under storage, and if so, what was the most efficacious concentration. Different groups of rabbit corneas were placed in saline as controls or in varying concentrations of hydrocortisone (10(-10) M to 10(-4) M at pH 7.4) at 37 degrees C and 4 degrees C. Acid phosphatase released after six hours was measured biochemically. This enzyme was used as a marker enzyme reflecting lysosomal labilization. Results showed a significant stabilization of the lysosomal membrane at 4 degrees C as compared to 37 degrees C. A trend towards stabilization of the lysosomal membrane was seen when 10(-8) M concentration of hydrocortisone at 37 degrees C was used, there being no demonstrable stabilization at 4 degrees C.

Enhancement and inhibition of phagocytic activity in the retinal pigment epithelium.

The phagocytic activity of the pigmented epithelium of the bovine retina was studied with an organ culture model. Latex particles coated with immunoglobulin, or bovine rod outer segments, both labelled with radioactive iodine, were the objects to be phagocytosed. Various agents were tested with this model for their effects on phagocytosis by the retinal pigment epithelium. Iodoacetate significantly inhibited phagocytosis, as did penicillin, though to a lesser extent. Concanavalin A and gentamicin enhanced phagocytosis. Dimethyl sulfoxide had no effect. The results with lymphokines were not conclusive.

Factors in the survival of stored corneas.

In studying the survival of stored corneas we wished to know if excising the cornea was the most critical step or if one storage medium was superior to another. Rabbit corneas were excised and stored in various media at 4 degrees C or 23 degrees C for four days. The storage media included M-K medium, balanced salt solution (BSS) and 10(-8)M concentration of hydrocortisone in M-K medium and BSS. Comparisons were made by measuring acid phosphatase as an index of autolysis. Results showed a reduced amount of autolysis at 4 degrees C as compared to 23 degrees C. No statistical difference between M-K medium and BSS was seen but steroid decreased autolysis. M-K medium plus hydrocortisone seemed to be the best of the solutions studied, but removal of the cornea seemed to be the most important factor in its survival.