Substance P and adrenocorticotropic hormone do not affect T-lymphocyte adhesion to vascular endothelium or surface expression of adhesion receptors

Int. J. lrnrnunopharmac., Vol. 16, No. 2, pp. 137-149, 1994 Elsevier Science Ltd Copyright © 1994 International Society for Immunopharmacology Printed in Great Britain. All rights reserved 0192-0561/94 $6.00 + .00

~ Pergamon

S U B S T A N C E P A N D A D R E N O C O R T I C O T R O P I C H O R M O N E DO NOT A F F E C T T - L Y M P H O C Y T E A D H E S I O N TO V A S C U L A R E N D O T H E L I U M OR S U R F A C E E X P R E S S I O N OF A D H E S I O N R E C E P T O R S BRIAN ALAN SMART, K. MURALI KRISHNA RAO* and HARVEY JAY COHEN Geriatric Research, Education, and Clinical Center, Veterans Administration Medical Center and Center for the Study of Aging and Human Development, Duke University Medical Center, Durham, NC 27705, U.S.A. (Received 24 February 1993 and in final form 16 August 1993)

Abstract - - Substance P (SP) and adrenocorticotropic hormone (ACTH) are peptides that have been shown to have both neurological and immunological effects. Because of the demonstrated effects upon immune function, we examined the effects of these peptides on T-lymphocyte adhesion to vascular endothelium and surface adhesion receptor expression. Neither the adhesion assays nor the expression assays showed any statistically significant effect of SP (10/aM) or ACTH (1 /JM) for any incubation period used. We conclude that, while SP and ACTH have a variety of immunomodulatory effects, direct modulation of T-lymphocyte adhesion to vascular endothelium is probably not one of them.

Substance P (SP) and adrenocorticotropic hormone (ACTH) are two peptides that are involved in stress responses. SP is a well characterized undecapeptide, widely distributed in the human central and peripheral nervous systems, that originally was found to be involved in nociceptive neurotransmission and other neurotransmitter functions (Pernow, 1983). ACTH is a peptide hormone produced by the pituitary that is known to induce adrenal production of the "stress hormone" hydrocortisone. Since the immune response is inherently associated with stress, these stress-related functions of SP and ACTH have led to studies which have found that both peptides have immunomodulatory functions. For example, SP has a number of effects upon lymphocytes including enhanced immunoglobulin synthesis and proliferation, both in vitro (McGillis, Organist & Payan, 1987; Beinenstock, Croitoru, Ernst, Stead & Stanisz, 1989) and in vivo (Scicchitano, Biennenstock & Stanisz, 1988). ACTH also has effects upon lymphocytes such as reduced immunoglobulin production (Carr, Radulescu, deCosta, Rice & Blalock, 1990) and increased growth and differentiation (Alvarez-Mon, Kehrl & Fauci, 1985). The cellular responses to SP and ACTH may be receptor-

mediated because receptors for both SP (Payan & Goetzl, 1987; Stanisz, Scicchitano, Dazin, Bienenstock & Payan, 1987; Greeno, Vercellotti, Mantyh, Maggio, Jacob & Moldow, 1991) and ACTH (Johnson, Klimpel & Smith, 1987; Johnson, Blalock & Smith, 1988; Wear, Bost, Smith & Blalock, 1987; Smith, Brosnan, Meyer & Blalock, 1987; Clarke & Bost, 1989) have been reported to be present on lymphocytes and other leukocytes. Because of the effects of SP and ACTH upon important lymphocyte functions, and because of the presence of receptors for these peptides, it is likely that there are other, undescribed, effects of SP and ACTH upon important aspects of lymphocyte function. One important aspect of lymphocyte function upon which the effects of SP and ACTH are largely unknown is lymphocyte adhesion to vascular endothelium. This adhesion process is essential for normal immune function because it is the first step in the extravasation process involved in inflammation and lymphocyte migration to peripheral lymphoid tissues (Springer, 1990). To our knowledge, no effects of SP or ACTH upon lymphocyte adhesion to vascular endothelium have been described, but SP has been shown to have some effects upon similar

*Author to whom correspondence should be addressed at: Bldg No. 10, Box 182 A, V. A. Medical Center, Durham, NC 27705, U.S.A. 137

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processes. For example, SP induces changes in neutrophil adhesion receptor expression, morphology, and migration (Shipp, Stefano, Switzer, Griffin & Reinherz, 1991), and induces a mast cellmediated increase in adhesion receptor expression by vascular endothelium (Matis, Lavker & Murphy, 1990). Therefore, since the effects of SP and ACTH upon lymphocyte adhesion to vascular endothelium have not been described, we have undertaken to measure these effects. Because the lymphocyte-endothelial cell adhesion process is mediated by a number of adhesion receptors that are expressed on lymphocytes (Springer, 1990), we measured the effects of SP and ACTH upon Tqymphocyte surface expression of the adhesion receptors, LAM-1 and LFA-I, along with T-lymphocyte adhesion to human umbilical vein endothelial cells (HUVEC). LAM-1 is a "selectin" that is also called L-Selectin and LECAM-1, while LFA-1 is in an "integrin" that is also called CD11a/CD18. We also measured the effects of SP and ACTH upon neutrophil expression of LAM-1 and CR3 (an adhesion receptor in the same subclass with LFA-I that is also called CD11b/CD18).

(solid) was obtained from the Fisher Scientific Company (Fair Lawn, N J). T-lymphocyte isolation

Blood was obtained, with informed consent, from peripheral veins of volunteers. Unless otherwise noted, all processing was performed at room temperature. Mononuclear cells were obtained from the blood using the F i c o l l - H y p a q u e density gradient technique (Boyum, 1968). The cells were washed twice in HBSS without calcium and magnesium and red blood cells were lysed with hypotonic shock. The mononuclear cells were then resuspended in RPMI-1640 containing 10% FCS and were twice depleted of monocytes and B-lymphocytes by adherence to plastic with 45-min incubations at 37°C (Oppenheimer-Marks, Davis & Lipsky, 1990; Doukas & Pober, 1990). Using this isolation technique, our mononuclear cells were found to be 96% T-lymphocytes with the anti-B/T-cell immunofluorescence kit (assessed by a FACScan technique similar to the technique described below). Neutrophil isolation

EXPERIMENTAL PROCEDURES

Materials

Substance P, ACTH, PMA, fetal calf serum (FCS) (heat inactivated), Rose Bengal, HEPES, sodium bicarbonate, heparin, dextran, and bovine serum albumin were obtained from Sigma (St Louis, MO). M199, RPMI-1640, phenol red-free Hanks' balanced salt solution (HBSS) with and without calcium (1 mM) and magnesium (0.5 mM), Dulbecco's phosphate-buffered saline (PBS), fungizone, penicillin-streptomycin (10,000 units/ml), and tryps i n - E D T A (10×) were obtained from Gibco (Grand Island, NY). Collagenase was obtained from the Worthington Biochemical Corporation (Freehold, N J). Anti-CD1 la, anti-Leu8, ar~ti B/T-cell kit, and goat anti-mouse Ig were obtained from BectonDickinson (San Jose, CA). A n t i - C D l l b was obtained from AMAC Inc. (Westbrook, ME). AntiFactor VIII was obtained from the Dako Corporation (Carpinteria, CA). FACS reference beads were obtained from the Flow Cytometry Standards Corporation (Research Triangle Park, NC). Endothelial cell growth supplement was obtained from Collaborative Research Incorporated (Bedford, MA). Tumor necrosis factor was obtained from R & D Systems (Minneapolis, MN). Paraformaldehyde

Heparinized (10 U/ml) blood was obtained, with informed consent, from peripheral veins of volunteers. All processing was performed at 4°C (on ice). A red cell pellet was obtained using the Ficoll - Hypaque density gradient technique (Boyum, 1968), and a dextran sedimentation technique was used to obtain neutrophils from the pellet (Rao, Currie, Padmanabhan & Cohen, 1992). The red cell pellet was diluted approximately 8-fold in HBSS and 4.5% dextran sulfate in phosphatebuffered saline (0.02 M, pH 7.4) was added in 1 : 5 ratio (dextran : pellet resuspension). The cells were allowed to settle for 45 min and the supernatant containing neutrophils was aspirated. The contaminating red cells were lysed with hypotonic shock and the neutrophils were washed twice in HBSS without calcium and magnesium. Finally, the neutrophils were suspended in HBSS containing calcium and magnesium for the experiments. Endothelial cell culture and preparation

Human umbilical vein endothelial cells (HUVEC) were obtained by a collagenase digestion technique in umbilical cord buffer (137 mM NaC1, 4 mM KCI, 10 mM HEPES, 11 mM glucose, pH 7.4) similar to a technique described previously (OppenheimerMarks et al., 1990). The HUVEC were then cultured in complete media (M-199 plus 16% FCS, 16 mM

Substance P and ACTH Effect on T-lymphocyte Adhesion

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HEPES, 0.02807o bicarbonate, fungizone, penicillin, streptomycin, endothelial cell growth supplement (100/ag/ml), and heparin (50 units/ml), pH 7.4) in gelatin-coated flasks at 37°C, 507o CO2 (Oppenheimer-Marks et al., 1990; Spertini et al., 1991). The HUVEC were removed from the flasks by trypsinization (Oppenheimer-Marks et al., 1990) and were passaged three to five times before finally being grown to confluent monolayers in 96-well plates for the adhesion assays. We used the progeny of a single cord for each plate. Factor VIII binding and immunofluorescence using goat anti-mouse Ig revealed the cultured monolayers to be endothelial cells (Gamble & Vadas, 1988).

A d h e s i o n assays Isolated T-lymphocytes and 96-well plates of endothelial cells were used for the adhesion assays. After washing the endothelial cells with M-199 plus 5°7o FCS, the endothelial cells were preincubated for 4 h with or without TNF, SP or ACTH at 37°C, 5°7o CO2. The T-lymphocytes in the 24-h incubation set were incubated for 24 h in RPMI-1640 plus 10070 FCS, penicillin, and streptomycin with or without PMA, SP, or ACTH; they were then washed and resuspended in M-199 plus 5070 FCS and added to the plates. The T-lymphocytes in the 45-min incubation set were resuspended after isolation in M-199 plus 5°7o FCS and were then added to the plates

140

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Fig. 1. Adherence of T-lymphocytes to HUVEC after a 45-min incubation. (A) Lymphocyte response to PMA and SP and endothelial cell response to TNF. (B) Lymphocyte response to SP and endothelial cell response to TNF and SP. (C) Lymphocyte response to ACTH and endothelial cell response to TNF and ACTH. (D) Lymphocyte response to SP, ACTH, PMA, and combinations of SP + PMA and ACTH + PMA. Because of the variability inherent in the small sample size in (D), values are expressed as the percent of maximum adherence for clarity. Reagent concentrations were as follows: SP, 10 juM; ACTH, 1 ~M; PMA, 100 nM; TNF, 100 units/ml. Bars: mean +_ S.E.M. of 13 - 15 (A), 6 (B), 11 (C), or 3 (D) separate experiments, each performed in triplicate. ** Statistically significant difference from control.

immediately after adding buffer, P M A , SP, A C T H , or c o m b i n a t i o n s o f these substances to the resuspensions. The T-lymphocyte resuspensions b o t h in the 24-h and 45-min assays were at a c o n c e n t r a t i o n range o f 1 . 5 - 5 x 106 cells/ml (counted with a Coulter cell counter, Hialeah, F L ) a n d 100tA o f resuspension was a d d e d per well. Both the 24-h and 45-min sets were incubated for 45 min at 37°C, 5°70 CO2 after the T-lymphocytes were added to the plates. The adherent T - l y m p h o cytes were then m e a s u r e d using a Rose Bengal

staining technique (Gamble & Vadas, 1988). Briefly, the cells were stained with a 0.25% Rose Bengal solution, washed four times with 37°C HBSS containing calcium and magnesium, and lysed with 50% ethanol before m e a s u r e m e n t with a 570 nm filter in a plate reader ( I C N / F l o w Laboratories Inc., McLean, VA). Blank values derived f r o m stained endothelial cells alone were subtracted and n u m b e r s o f adherent T-lymphocytes were determined using s t a n d a r d curves o f counted, stained T-lymphocytes.

Substance P and ACTH Effect on T-lymphocyte Adhesion

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Fig. 2. Adherence of T-lymphocytes to HUVEC after a 24-h incubation. Lymphocytes were treated with PMA, SP, or ACTH for 24 h and endothelial cells were treated with TNF, SP, or ACTH for 4 h before the lymphocytes were added to the HUVEC. Reagent concentrations were as follows: SP, 10 ~M; ACTH, I /aM; PMA, 100 nM; TNF, 100 units/ml. Bars: mean __+_S.E.M. of 6 - 8 separate experiments, each performed in triplicate. +÷ Value approaching statistical difference from control.

Adhesion receptor expression assays T-lymphocyte assays. T-lymphocytes

were resuspended at a concentration of 1 × 106 cells/ml in RPMI-1640 plus 10%0 FCS, penicillin, and streptomycin. The cells were then incubated for either 45 min or 24 h at 37°C, 5% COz with buffer, PMA, SP, or A C T H . After incubation, the cells were washed twice with PBS and were resuspended in 100/A of PBS at a concentration of 5 × 106 cells/ml. The cells were then labelled with either 10/al of fluoresceinated a n t i - C D l l a (LFA-1) or 4/al of fluoresceinated anti-Leu8 (LAM-1) and were incubated at 4°C for 30 min, washed twice with PBS, and fixed in a PBS solution containing 1% paraformaldehyde and 0.05% bovine serum albumin (Currie, Rao, Padmanabhan, Jones, Crawford & Cohen, 1990). The fixed cells were then analyzed using a FACScan (Becton-Dickinson, San Jose, CA) calibrated with FACS reference beads (Rao, Currie, Ruff & Cohen, 1988).

Neutrophil assays Neutrophils were resuspended at a concentration of 10 × 106 cells/ml in HBSS containing calcium and magnesium. The cells were then incubated for 10 min at 37°C with or without PMA, SP, or ACTH. After the incubation, either 8/A of anti-Leu8 (LAM-I) or 4/al of flouresceinated a n t i - C D l l b (CR3) was added to each tube. The cells were then

incubated on ice for an additional 30 min. After this incubation, the cells were washed with cold PBS (1 min spin in a microfuge), and fixed in 100/A of 1%0 paraformaldehyde and 0.05% bovine serum albumin. The fixed cells were then analyzed using a FACScan calibrated with FACS reference beads.

Statistical analysb Paired, two-tailed Student's t-tests were used to analyze data.

RESULTS

T-lymphocyte adhesion to endothelial cells The adhesion assays were performed for 45-min and 24-h incubation periods. Incubation of lymphocytes for 45 min with SP [Figs I(A), I(B)] or with A C T H [Fig. I(C)] did not significantly alter T-lymphocyte adhesion to HUVEC (/>0.05). Preincubation of endothelial cells for 4 h with SP [Fig. I(B)] or with A C T H [Fig. I(C)] also did not significantly alter lymphocyte adhesion to HUVEC (P>0.05). In contrast, lymphocytes incubated for 45 min with PMA, and H U V EC pre-incubated for 4 h with TNF, showed significantly greater adhesion with their targets (P<0.0005). However, when the lymphocytes were incubated for 45 min with P MA + SP or PMA + A C T H [Fig. I(D)], there

B . A . SMART et al.

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Fig. 3. T-lymphocyte surface expression of adhesion receptors after treatment with SP, ACTH, and PMA. (A) 45-rain incubation. (B) 24-h incubation. Values represent the ratio of sample mean channel fluorescence to control mean channel fluorescence as determined by FACS analysis. Reagent concentrations were as follows: SP, 10/aM; ACTH, 1 ~M; PMA, 100 nM. Bars: mean _+ S.E.M. of 5 - 8 (A) or 6 - 8 (B) separate experiments. **Statistically significant difference from control.

was n o significant change (P>0.05) f r o m the increased levels o f a d h e s i o n t h a t result f r o m P M A s t i m u l a t i o n alone. T h e 24-h i n c u b a t i o n a d h e s i o n assay results were generally similar to those o f the 45-min i n c u b a t i o n a d h e s i o n assays. T - l y m p h o c y t e i n c u b a t i o n for 24 h with SP or A C T H a n d endothelial cell prei n c u b a t i o n for 4 h with SP or A C T H (Fig. 2) did not significantly alter a d h e s i o n o f lymphocytes to H U V E C (P>0.05). T h e r e were, however, some differences between the 24-h a n d 45-rain i n c u b a t i o n data. In the 24-h i n c u b a t i o n experiments, P M A

treatment of lymphocytes (Fig. 2) significantly alter a d h e s i o n (P>0.05) HUVEC p r e - i n c u b a t i o n with T N F a d h e s i o n a n d a p p r o a c h e d , but did not significance ( P = 0.0503).

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Expression assays for LFA-1 a n d LAM-1 were p e r f o r m e d for 45-min a n d 24-h i n c u b a t i o n s . Similar to the a d h e s i o n experiments, neither the 45-min i n c u b a t i o n [Fig. 3(A)] n o r the 24-h i n c u b a t i o n

Substance P and ACTH Effect on T-lymphocyte Adhesion

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Fig. 4. Dose response of surface adhesion receptor expression to a 45-min incubation with SP (A) and ACTH (B). Values represent the ratio of sample mean channel fluorescence to control mean channel fluorescence as determined by FACS analysis. Data are from two experiments, with the exception of 10/aM SP in (A) (five experiments) and 1 /~M ACTH in (B) (five experiments). Bars: mean _+ S.E.M. of values for 10/aM SP (A) and 1 /aM ACTH (B).

[Fig. 3(B)] with SP or A C T H significantly altered the cell surface expression of LFA-1 or LAM-1 (P>0.05). P MA did not significantly affect LFA-1 expression for either incubation period (P>0.05), while it induced significant reduction of LAM-I levels by 45 rain (/'<0.0005). The reduction of LAM-I at 24 h of incubation with P M A was also significant (P<0.05), but was of smaller magnitude. Dose response studies of SP and A C T H on LFA-1 and LAM-1 expression were performed for 45 rain [Figs4(A), 4(B)] and 2 4 h [Figs 5(A), 5(B)]. After both incubation periods, SP and A C T H were

found to have minimal effects upon LFA-1 and LAM-1 expression for their entire dose ranges (100/~M- 10 nM for SP, 2 / ~ M - 1 nM for ACTH).

Neutrophil surface expression of adhesion receptors Expression assays for LAM-I and CR3 were performed with neutrophils for 10-min incubations with SP and ACTH. SP was found to significantly decrease neutrophil expression of LAM-1 (P<0.05) [Fig. 6(A)] and to significantly increase neutrophil expression of CR3 (P<0.05) [Fig. 6(B)]. A C T H was

144

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Fig. 5. Dose response of surface adhesion receptor expression to a 24-h incubation with SP (A) and ACTH (B). Values represent the ratio of sample mean channel fluorescence to control mean channel fluorescence as determined by FACS analysis. Data are from two experiments in (A) and three experiments in (B), with the exception of 10 ~M SP in (A) (eight experiments) and 1 ~M ACTH in (B) (six experiments). Bars: mean _+ S.E.M. of values for 10 gM SP (A) and 1 ~M ACTH (B). found to have no significant effect upon neutrophil expression of either LAM-1 or CR3 (results not shown).

DISCUSSION The peptides SP and A C T H have many welldescribed effects upon immune function. For example, beside the effects of SP mentioned previously, upon lymphocyte immunoglobulin production and proliferation, this peptide has been

shown to be involved in neurogenic inflammation (Pernow, 1985), psoriasis (Koblenzer, 1987), vascular permeability changes (Lundberg, Saria, Brodin, Rosell & Folkers, 1983), immediate hypersensitivity (Goetzl, Chernov, Renold & Payan, 1985), and the dermatological flare and itch (Hagermark, Hokfelt & Pernow, 1978). Specific cellular effects of SP include mast cell release of histamine (Shanahan, Denburg, Fox, Bienenstock & Befus, 1985), induction of neutrophil cytotoxic activity (Wozniak, McLennan, Botts, Murphy & Scicchitano, 1989), monocyte release of inflammatory cytokines

Substance P and ACTH Effect on T-lymphocyte Adhesion

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Fig. 6. Neutrophil surface expression of LAM-1 (A) or CR3 (B) with or without treatment with SP. Values represent the average sample mean channel fluorescence as determined by FACS analysis. The reagent concentration was as follows: SP, 10 ~M. Bars: mean _ S.E.M. of four separate experiments. **Statistically significant difference from control. (Lotz, Vaughan & Carson, 1988), and enhanced IL-2 expression in activated T-lymphocytes (Calvo, Chavanel & Senik, 1992). A C T H has also been shown to have a number of effects beside those, mentioned previously, upon lymphocyte immunoglobulin production and proliferation. For example, A C T H blocks y-interferon production by spleen cell cultures (Johnson, Torres, Smith, Dion & Blalock, 1984), reduces the in vitro antibody response to both T-cell dependent and independent antigens (Johnson, Smith, Torres & Blalock, 1982), and enhances memory cytotoxic activity (Johnson et al., 1987). However, despite the wide range of reported effects of SP and A C T H upon immune function, our

study has shown no statistically significant effect of these peptides upon T-lymphocyte adhesion to vascular endothelium or T-lymphocyte expression of the adhesion receptors LFA-1 and LAM-1 in vitro. This absence of effect was found for both 45-min and 24-h incubations with SP and ACTH. Furthermore, in the adherence studies, the combination of SP or A C T H with PMA or preincubation of HUVEC with SP or A C T H had no effect. In contrast to SP and A CTH , the positive controls PMA and TNF generally had their anticipated effects. For example, similar to previous studies, when T-lymphocytes were incubated for 45 min with PMA, and H U V EC were pre-incubated for 4 h with

146

B. A. SMARTet al.

TNF, there was significantly greater adhesion (Dustin & Springer, 1989; Mantovani, Bussolino & Dejana, 1992; Shimizu et al., 1991; Springer, 1990). Also consistent with previously published results, PMA did not significantly affect LFA-1 expression for either incubation period (Dustin & Springer, 1989), while PMA induced significant reduction of LAM-1 levels by 45 min (Jung & Dailey, 1990). An unanticipated result, however, was that 24-h incubation of T-lymphocytes with PMA did not significantly alter adhesion. This lack of effect for PMA is in contrast to previously published results in which another phorbol ester (phorbol dibutyrate) increased adherence at 24 h of incubation but not after 48 h (Openheimer-Marks et al., 1990). This contrasting result may be explained by biochemical differences between the two phorbol esters (different binding efficiency to PKC, for example) resulting in a less sustained response in our experiments. The absence of an effect of PMA upon adhesion at 24 h of incubation may be consistent with the relative increase in LAM-1 expression at 24 h of incubation with PMA as compared to 45 min of incubation because it may indicate that PMA could not sustain a reduction of LAM-1 expression for this length of time, just as it could not sustain an increase in adhesion. Another unexpected result with the 24-h lymphocyte incubations is that the 4-h HUVEC preincubation with TNF approached, but did not achieve a significant increase in adhesion. Since TNF primarily increases adhesiveness of HUVEC to activated lymphocytes through the adhesion receptor ICAM-1, the primary ligand for LFA-1 (Shimizu et al., 1991), and since culture of lymphocytes in the presence of fetal calf serum (as we have done) for as little as 2 h activates them and promotes increased adhesion of lymphocytes to HUVEC through LFA-1 (Oppenheimer-Marks et al., 1990), it is possible that, similar to the PMA results both with adhesion and LAM-1 expression, lymphocyte activation could not be sustained for 24 h and LFA-1 could no longer bind ICAM-1 with as much avidity. Therefore, increased ICAM-1 would have a blunted effect and statistically significant change would be more difficult to demonstrate. Thus, despite the slight differences between previously reported results and our 24-h incubation experiments, PMA and TNF did generally have their expected effects. Though the absence of effects for SP and ACTH could be due to design or technical considerations, we do not believe this to be the case. For example, because of the high concentrations of SP (10 ~M) and ACTH (1 ~M) used in this study and because of

the range surveyed by the dose response experiments (SP: 1 0 0 ~ M - 1 0 n M and ACTH: 2 ~ M - 1 nM), any effect of SP or ACTH should have been found if it existed. Furthermore, in the neutrophil adhesion receptor expression experiments, SP was found to decrease levels of LAM-1 and increase levels of CR3, similar to previously reported results (Shipp et al., 1991), indicating that the SP used in these experiments was biologically active. ACTH was not found to have effects upon neutrophil adhesion receptor expression, but we do not know of any previous study in which ACTH has affected neutrophil adhesion receptor expression. In addition, since PMA and TNF generally had their expected effects, it is unlikely that technical errors played a role in the absence of effect for SP and ACTH. SP and ACTH did not affect overall T-lymphocyte adhesion or LFA-1 and LAM-1 expression, but adhesion phenomena are known to be regulated in more ways than just the total lymphocyte surface expression of these two receptors. For instance, both LFA-1 and LAM-1 function are regulated both on the levels of quantity of cell surface expression and of avidity of interaction (Dustin & Springer, 1991; Figdor, Van Kooyk & Keizer, 1990; Inghirami, Grignani, Sternas, Lombardi, Knowles & DallaFavera, 1990; Jung & Dailey, 1990; Kansas, Spertini, Stoolman & Tedder, 1991; Spertini, Kansas, Munro, Griffin & Tedder, 1991). Therefore, our study was designed to determine total cell - cell adhesion and to correlate this with receptor expression. We could thereby assess changes in receptor avidity that could compensate for changes in receptor numbers. For example, our PMA results show a significant increase in adhesion with 45 min of incubation, but no change in LFA-1 expression. These results are consistent with published results that have shown that there is an increase in LFA-1 avidity in response to PMA (Dustin & Springer, 1989). The SP and ACTH results therefore imply that there is no LFA-I or LAM-1 adhesion receptor avidity change to compensate for the lack of change in receptor expression levels. In addition to changes in lymphocyte LFA-1 and LAM-1 expression and avidity, adhesion phenomena are regulated by the utilization of different receptors by both lymphocytes and HUVEC during different activation states. In lymphocytes, for example, a number of other adhesion receptors have been described beside LFA-I and LAM-I (Dustin & Springer, 1991; Shimizu et al., 1991; Stoolman, 1989; Springer, 1990), but LFA-1 is the primary adhesion receptor used within minutes of incubation

Substance P and ACTH Effect on T-lymphocyte Adhesion with PMA (Shimizu et al., 1991). Adhesive interactions are also regulated at the level of the endothelial cell (Mantovani et al., 1992; Pober & Cotran, 1991). After endothelial cells have been activated with 4 h of cytokine treatment (with TNF or IL-1, for example), adherence to resting lymphocytes is primarily mediated through the endothelial cell adhesion receptor, ELAM-1 (Shimizu et al., 1991), and adherence to PMAactivated lymphocytes is primarily mediated through the endothelial cell adhesion receptor, ICAM-1, the primary ligand for LFA-1 (Shimizu et al., 1991). Four hours of cytokine incubation with endothelial cells significantly increases ICAM-1 expression (Shimizu et al., 1991), while lymphocyte incubation with PMA increases LFA-1 avidity (Dustin & Springer, 1989), thus accounting for the additive effect of endothelial cell stimulation with TNF and lymphocyte incubation with PMA. Since lymphocyte LFA-1 and endothelial cell ELAM-1 and ICAM-1 are the most important receptors in the different combinations of cellular activated states outlined above, and since SP and ACTH do not increase lymphocyte adhesiveness or endothelial cell adhesiveness, or demonstrate an additive effect upon incubation with both cell types, it is unlikely that these peptides increase the function of these specific adhesion receptors. While we have concluded that SP and ACTH do not affect T-lymphocyte adhesion and are unlikely to affect the function of certain specific receptors, we can also comment upon the signal transduction mechanisms of the lymphocyte SP and ACTH receptors. A number of studies have indicated that protein kinase C (PKC) function may be affected by SP (Catalan, Martinez, Aragones & Fernandez, 1989; Holzer & Lippe, 1989) and ACTH (Cozza, Vila, Acevedo-Duncan, Farese & Gomez-Sanchez, 1990; Cozza, Vila, Acevedo-Duncan, GomezSanchez & Farese, 1990; Lehoux, Grondin, Pacuraru & Yachaoui, 1991) in various non-lymphocyte cell types. However, our study indicates that, in T-lymphocytes, SP and ACTH are unlikely to affect PKC because of differences between the effects of these peptides and of PMA, which is an analog of

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diacyclycerol that can directly stimulate PKC (Figdor, Van Kooyk & Keizer, 1990). Stimulation with PMA activates LFA-1 (Rothlein & Springer, 1986), thus increasing adhesion (Figdor et al., 1990), and induces shedding of LAM-1 (Jung & Dailey, 1990). The absence of these effects in response to SP and ACTH stimulation implies that, in T-lymphocytes, the SP and ACTH receptors do not function through PKC and lends more importance to the report that, in mononuclear cells, ACTH has been shown to increase cyclic AMP levels (Johnson et al., 1988). However, it is also possible that SP and ACTH might affect PKC, but that this phenomenon is masked in our study by a process such as inhibition of another element of the signal transduction mechanism. The absence of in vitro effects for SP or ACTH, reported here, does not, however, preclude the possibility of in vivo roles of SP or ACTH since these peptides could have indirect effects. For example, as previously mentioned, SP has been reported to increase adhesion receptor expression on vascular endothelium indirectly via a mast cell-mediated process (Matis et al., 1990). ACTH could have similar indirect effects upon the lymphocyteendothelial cell adhesion process. Clearly, an in vitro study such as ours cannot measure the wide range of in vivo effects that SP and ACTH could have upon lymphocyte adhesion to vascular endothelium. However, we can conclude with some degree of confidence that SP and ACTH do not have physiologically relevant direct effects upon T-lymphocyte adhesion to vascular endothelium. Acknowledgements - - We would like to thank Drs Mark S.

Currie and Richard Levenson for their invaluable advice and encouragement and Diana Kilby and Celeste Copeland for their superb technical assistance. We would also like to thank Ms Sandra Wilkins and the nursing staff of the obstetrics unit at Durham Regional Hospital for generously supplying us with umbilical cords. B. A. S. was supported by a Hartford/Stead research scholarship for third year medical students funded by a John A. Hartford Foundation grant to Duke University for the Academic Geriatric Recruitment Initiative. This study was supported by the VA Medical Research Service.

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