I Ill I I Ill Ill I III I I II I I I l l

General discussion of articles by Brinckerhoff et al, Bauer et al, De Luca, Camisa et al, and McGuire et al

A. R. Shalita, M.D.: Dr. Camisa, you seem to be saying that whatever factors effect neutrophil chemotaxis may be releasing lysosomal enzymes and this becomes a self-perpetuating mechanism for con- tinued chemotaxis attracting other neutrophils into the area. With that hypothesis, how would you explain the action of etretinate? In the histologic sections that you showed from psoriatic patients treated with etreti- nate, the neutrophils disappeared after treatment with etretinate.

C. Camisa, M.D.: We have also been looking at biopsies from patients with psoriasis treated with other modalities such as UVB and methotrexate. With those two modalities we found that the neutrophils remained in the lesions longer than in those lesions treated with etretinate for a comparable length of time. However, this was not the case for topical corticosteroids. I am really unable to answer your question except to say that similar histopathologic changes have been seen in pso- riatic lesions treated with topical corticosteroids, and more biopsies are being examined to further clarify this issue.

A. R. Shalita, M.D.: You didn't show an effect on chemotaxis and chemokinesis. Is it true that movement of the cells was not affected?

C. Camisa, M.D.: That is correct for the concentra- tions that were studied, which ranged from 10 -6 to 10 -~.

A. R. Shalita, M.D.: There is an effect on chemo- taxis; the problem is that it has occurred only with cytotoxic doses of the drug, so it is not a real life kind of situation.

C, Camisa, M.D.: Have you found that, Dr. Shalita?

A, R. Shalita, M.D.: Yes. C, Camisa, M.D.: At what concentration? A. R. Shalita, M.D.: 10 -z and 10 -2 G. G. Krueger, M.D.: First a comment and then

two questions. The comment is related t~ the number of microabscesses that one sees if one does serial sections through a biopsy of psoriasis. Last summer Russ Eyre, a medical student, spent considerable time in my labo- ratory looking at serial sections of biopsies taken from

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eighteen psoriatic patients. In those eighteen serially sectioned biopsies, we examined a 200 × 50-~z area for Munro microabscesses. We found fourteen microab- scesses; twelve of them were in one patient. The other two were in other patients. The bottom line at this time is that, although the neutrophil is there and pustular psoriasis does exist, I think we are in some difficulty in trying to link psoriasis and the neutrophil.

C. Camisa , M.D.: My reply to your comment is that the tentative conclusion of this study is that the anti-inflammatory effects of etretinate are weak, and this effect is probably not a factor in the resolution of psoriatic lesions,

G. G. Krueger , M.D.: My comment before Dr. Shalita said anything was going to be that I think that Dr. Camisa clearly showed that the neutrophil does not have anything to do with psoriasis or that at least etretinate has nothing to do with neutrophil function. But, again I am not willing to commit myse l f on that. The question I have is related to the serum factor. Are you suggesting that perhaps there are proteins that bind vitamin A or the synthetic products we are talking about here and they have no physiologic function; i.e., if you put serum in the system you would anticipate that this presents carrier proteins that should allow delivery to the intracellular contents? Thus you should have en- hanced, I would think, most of the activities; instead, you suppressed them all. I wonder how you address that question?

C. Camisa , M.D.: I addressed it in the same way that you concluded; I thought that there was a factor in the serum (probably albumin) that bound the retinoid and prevented it from associating with the cell. My conclusion was that free retinoid was most active in these in vitro experiments. I have been able to quanti- tate the amount ofretinoid associated with the cells by a very simple technic which shows that the amount of inhibition of lysosomal enzyme release is directly re- lated to the concentration of retinoid associated with the polymorphonuclear leukocytes, whether serum is ab- sent or present.

G. G. Krueger , M.D.: You are basically saying

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Volume 6 Number 4, Part 2 April, 1982

General discussion 641

that you can bypass the serum protein system and show intracellular effects.

C. Camisa, M.D.: Yes. For example, when a final concentration of 20/zmoles tretinoin is added to cells without serum, I get 50% inhibition of/3-glucuronidase release. When that same final concentration of tretinoin (20 moles) is added to cells in the presence of 10% serum, no inhibition of enzyme release is seen. When I extract the ceils as described above, the retinoid is bound to the cells in the first example, and none is bound to the cells in the second case, because it is free in the supernatant, presumably bound to carrier mole- cules in the serum.

J. J. Voorhees, M.D.: Dr. Camisa, you showed effects in vitro but you were showing basically no ef- fects in vivo. It seems to me that since the concentra- tion of the drug, etretinate, for example, in vivo is about 0.1 to 0.5/xg, you were operating at greater than one hundred-fold that concentration in vitro. It seems to me that the data can be explained conceivably on that basis alone.

C. Camisa, M.D.: The actual concentrations of these drugs in the skin have not really been established. Concentrations of drugs there may actually be higher than in blood.

J. J. Voorhees, M.D.: That is correct as a general rule. Generally speaking, drug concentrations are higher in the serum than they are in a tissue unless the tissue in question concentrates the drug.

C. Camisa, M.D.: Well, that is the point; the keratinocytes or neutrophils may concentrate the drug in vivo. The other point that I would like to make is that when topical tretinoin is used for the treatment of ache, it is applied in concentrations that approach 1 mmole; the 0.1% concentration is approximately 1 mmole or 10 -3, which is a hundred times higher than the concen- trations at which I have seen an inhibitory effect on enzyme release, but I still do not know the concentra- tion of that drug in the epidermis.

E. A. Bauer, M.D.: I am emban'assed to say I do not remember the specific data, but the group at Duke looked at that very question in patients with Darier's disease being treated with isotretinoin. They looked at lysosomal enzymes in the skin as they responded to treatment.

L. A. Goldsmith, M.D.: In our studies of epidermal skin biopsies, which were reported last year U Invest Dermatol 75:133, 1980), neutral proteinase did not change; lysosomal cathepsin D and /3-glucuronidase decreased. The patients with Darier's disease started with low levels of neutral proteinase, and with therapy those enzymatic activities increased.

E. A. Bauer, M.D.: Yes, some of them went down,

but there certainly was no increase. There was some inhibition.

J, J . Voorhees~ M.D.: I don't understand how more cornified envelopes can be present at the same time the cultures are blocked at a particular mode of differentia- tion with retinoid?

J. S. McGuire, M.D.: Several events occur in these maturing cultures, and the retinoids may influence one or more of them. We were surprised to find more en- velope formation with retinoids, but the finding was consistent.

K. M. Halprin, M.D.: Normally cells are pro- grammed to form the cornified envelopes before they slough off. So it should not be hard to conceive of that happening in the culture.

T. L. Ray, M.D.: Two questions and a comment. Dr. De Luca, your studies show that bovine serum albumin (BSA) is required for the transfer of mannose in your system. Do you know if retinoid-binding pro- tein (RBP) is in the fraction you used, and, if so, does it participate in this reaction'?.

L. M. De Luea, Ph.D.- Yes, this is an interesting possibility. We haven't really dissected it out. We found the effect with BSA; it was a very good effect and carried on with it. We don't know whether RBP may be more effective than BSA as a carrier for retinyl- phosphate.

T. L. Ray, M.D.: Your studies eloquently show the transfer of mannose. My second question is, "What other sugars can you transfer in this system?"

L. M. De Luca, Ph.D.: No other sugars can be transferred as far as we can tell. We have looked at glucosamine, galactosamine, glucose, fucose, and ga- lactose, and these are not stimulated in this system, so it appears to be a very specific system. I should state that we have not looked at xylose. There are some reports that the retinylphosphate xylose may also be formed.

T. R. Ray, M.D.: Finally, a comment regarding the neutrophil. There are in vitro studies showing that neu- trophil and phagocyte proteases can cleave comple- ment components and release C5a chemotactic activity. There are also studies showing that leukocyte proteases (elastase and cathepsin G) will inactivate chemotaetic factors, including CSa. These studies use purified sub- strate systems. Whether either of these reactions occurs in vivo is uncertain, since the addition of other protein substrates, such as albumin, will abrogate these reac- tions (J Clin lnvest 60:I280, 1977). These proteases can hydrolyze diverse proteins, yet their specificity for complement may be low and inhibitable by many alter- native protein substrates in the in vivo milieu. Thus, the augmentation of neutrophil accumulation via neutrophil

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activation of complement may not operate in the in vivo setting.

J. S. McGuire, M.D.: Ian King et al recently ob- served (Biochem J 194:341-350, 1981) that retinoids stimulate the incorporation of glucosamine into cell surface-associated hyaluronic acid. I don't know if this related to transport. There may be more than one site of action.

L. M. De Luca, Ph.D.: I would like to comment that Dr. Ray's question was more as to whether or not retinylphosphate sugar compounds other than retinyl- phosphomannose are formed, and I have responded to that; in other words, is there any other sugar attached to the retinylphosphate? My answer is that we haven't found any other sugar. Now in terms of stimulation of sugar incorporation, there will be a stimulation by treti- noin, depending on the system, and many such studies have been reported, but these could be indirect effects.

K. M. Halprin, M.D.: Do you think it could all be one defect causing everything else? Could the mannose transfer be basic?

L. M. De Luca, Ph.D.: i don't know at this time. I really don't know, but that is a consistent observation that we have.

L. A. Goldsmith, M.D.: I think it is useful to try to use Occam's razor on a very complicated epidermal cell system in culture. Your stained culture dishes sug- gested that growth was really decreased. One of my questions is whether there are some direct measure- ments of growth? Now, if growth is down and if protein synthesis is inhibited, I think there will be increased cell envelopes. I believe there is a real relationship between decreased growth and increased terminal dif- ferentiation,

J. S. McGuire, M.D.: The cultures that I showed you initially were not a steady-state system, and several things are happening: adhesion, cell movement, cell di vision, and dishesion of cells from the culture into the median. People who have tried to study the effects of retinoids in that kind of system observe various phe- nomena which are very difficult to put together into a convincing story. One can dissect the system by look- ing at the influence of retinoids on each of these pro- cesses. The inhibition of growth by retinoid in growing cultures cannot be compared with the influence of reti- noids on confluent or steady-state cultures in which

there is a tremendous acceleration in the number of cells that are shed. If shedding is somehow associated with increased envelope formation in these cultures, that is fine.

K. M. t la lprin, M.D.: When these cultures are in the steady-state, are they also inhibited in growth by the retinoids?

J. S. McGuire, M.D.: There is less protein attached to the dish after 3 weeks of exposure to retinoids.

F. L. Meyskens, Jr.~ M.D.: This is directed to Dr. De Luca. A lot of mitogenic and antimitogenic re- sponses appear to be modulated by membrane-bound receptors in response to epidermal growth factor (EGF), sarcoma growth factor (SGF), and similar ma- terials. Is it known whether the surface area that is most modified by vitamin A is involved at all in any of these or related receptors?

L. M, De Luca, Ph.D.: Yes. In fact, in our own laboratory, Dr. Jetten has shown that EGF receptor is increased seven- to tenfold upon exposure of cells to retinoids, We have tried about twenty different cell lines, and they all respond that way. So there is a specific increase in EGF receptor. But if you look at the insulin receptor, for instance, the increase is only about 1.2-fold over control; and the increase in concanavalin A receptor activity is very little--about twofold. So, there seems to be some specificity, and I think probably that may depend on the cell line.

F. L. Meyskens, Jr . , M.D.: The EGF response could be an indirect effect. It could be possible to look directly, using labeled mannose and labeled EGF, to see if it is the same area that is being affected, thereby representing actual structural changes on the membrane receptors.

L. M. De Luca, Ph.D.: It is possible, but it has not been done.

B. A. Pawson~ Ph.D.: Recently, leukotriene B 4, a product of metabolism of arachidonic acid by the lip- oxygenase pathway, has been described as a potent chemotactic factor and has been implicated as a media- tor in the inflammatory process. I am wondering if any of you have looked at the effect of retinoids on the formation of, or the action, of this substance?

C. Camisa, M.D.: I have not, but that is something that could be done in Dr. Weissmann's laboratory.