Photofrin ®, but not benzoporphyrin derivative, stimulates hematopoiesis in the mouse

Photofrin ®, but not benzoporphyrin derivative, stimulates hematopoiesis in the mouse

Immunopharmacology, 26 (1993) 203-212 © 1993 Elsevier Science Publishers B.V. All rights reserved 0162-3109/93/$06.00 203 I M P H A R 00664 Photofr...

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Immunopharmacology, 26 (1993) 203-212 © 1993 Elsevier Science Publishers B.V. All rights reserved 0162-3109/93/$06.00

203

I M P H A R 00664

Photofrin®, but not benzoporphyrin derivative, stimulates hematopoiesis in the mouse David W.C. H u n t a, Robert A. Sorrenti b, Claire B. Smits b and Julia G. Levy a aDepartrnent of Microbiology, University of British Columbia, Vancouver, BC, Canada V6T 1 WS and b Quadra Logic Technologies, Vancouver, BC, Canada (Received 30 December 1992; accepted 26 April 1993)

This report describes the effects of the porphyrin photosensitizers, Photofrin ® and benzoporphyrin derivative (BPD) on the immunohematopoietic system of normal and immunosuppressed DBA/2 mice in the absence of activating light. Photofrin ® (10 and 25 mg/kg) significantly increased in vitro colony formation by cells of the granulocyte-macrophage lineage in the spleen and bone marrow. Splenic hypercellularity, splenomegaly and elevated levels of blood leukocytes were observed in these mice 7 days following Photofrin ® injection. Evidence that Photofrin ® influenced the lymphohematopoietic compartment was suggested by a significant increase in blood lymphocytes and a population of spleen cells identified by a monoclonal antibody (LR- 1) reactive with mouse splenic B lymphocytes. Proliferative reponses of spleen cells from Photofrin®-treated mice to sub-optimal concentrations of Con A were greater than that observed for controls. However, spleen cell responses to LPS were unaltered by Photoffin ® administration. In contrast, BPD (10 mg/kg) did not alter any of the immunohematopoietic parameters studied. When Photofrin ® was administered to mice treated with the myeloablative agent 5-FU there was a significant acceleration in the recovery of total blood leukocyte and spleen cell numbers, relative to the controls. These studies demonstrate that, in addition to its previously documented activities as a photosensitizer, Photofrin ® can exert stimulatory effects upon murine hematopoiesis. Abstract:

Key words"

Porphyrin; Dihematoporphyrin ether; Benzoporpyrin derivative; Hematopoiesis; Photodynamic therapy

Introduction Porphyrins are a class of tetrapyrrolic compounds that are active in a wide variety of biological processes. Certain naturally occurring

Correspondence to: J.G. Levy, Department of Microbiology, University of British Columbia, Vancouver, Canada V6T 1W5. Abbreviations: CFU-GM, colony forming units granulocytemacrophage; Con A, concanavalin A; FITC, fluorosceinisothiocyanate; 5-FU, 5-fluorouracil; i.p., intraperitoneal; i.v., intravenous; lps, lipopolysaccharide A; PBS, phosphatebuffered saline.

and synthetic porphyrin molecules exhibit tumorlocalizing and strong light absorbance properties. These features have been utilized to elicit tumor destruction by the generation of toxic oxygen products when the tissue is exposed to visible light and constitutes the basis of photodynamic therapy (Dougherty, 1984). Despite intensive research on the photodynamic treatment of malignant tumors, the influence of photosensitizing porphyrins on normal tissues has largely been overlooked. However, studies by Canti et al. (1984, 1989) indicated that hematoporphyrin and Photofrin ® could accelerate the recovery of certain hematopoietic indices including spleen

204 weight, and spleen and bone marrow (BM) cell numbers of mice exposed to sub-lethal doses of 7-radiation or cytotoxic drugs. The cellular mechanisms by which porphyrins influence hematopoiesis are currently unknown. Protoporphyrin IX and related porphyrin analogues are activators of soluble guanylate cyclase (Ignarro et al., 1984), an enzyme which catalyzes the conversion ofguanosine 5'-triphosphate to cyclic guanosine monophosphate (cGMP). c G M P acts as an intracellular second messenger in a variety of biochemical pathways (Ignarro, 1989), although little is known of its role in the regulation of hematopoietic stem cell activity. Heroin (Stenzel et al., 1981) and metalloporphyrins (Novogrodsky, 1989), although not photosenstizers, are mitogenic for human T lymphocytes. In the present work we have examined the effects of Photofrin ® or BPD upon mouse myeloid lineage progenitor cells and functional responses of cells of the lymphoid lineage following the administration of Photofrin ® or BPD. Both of these compounds are being evaluated in human clinical trials for the photodynamic treatment of malignancies.

Materials and Methods

Animals Male DBA/2 mice of 8-12 weeks of age, obtained from Charles River (Montreal, Que.), were maintained under conditions of 12 h light/12 h dark. Animals were housed in plastic cages and provided standard laboratory chow and acidified water ad libitum. Porphyrin preparations Clinical-grade, sterile, pyrogen-free Photofrin ® (dihematoporpyrin ether) was provided by Quadra Logic Technologies (Vancouver, B.C.). Benzoporphyrin derivative mono-acid ring A (BPD) was synthesized as described (Pangka et al. 1986) and also provided by Quadra Logic Technologies. Porphyrin preparations were manipulated under low light conditions.

Experimental protocol Photofrin ® was reconstituted (2.5 mg/ml) in pyrogen-free 5j°o dextrose (Baxter Corp., Toronto, Ont.) and given i.p. or i.v. BPD was dissolved in 100°6 tissue culture grade dimethyl sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) at 20 mg/ml, diluted with PBS (pH7.3) immediately before use and injected i.v. Control animals received the corresponding solvent. Animals were kept in the dark for 24 h following drug administration. Mice were sacrificed by overdosing with Halothane ® (M.T.C. Pharmaceuticals, Cambridge, Ont.), blood was collected via cardiac puncture and hematocrit measurements performed. Blood smears were prepared, stained with the Wright-Giemsa reagent (Fisher Scientific, Vancouver, B.C.) and examined microscopically to obtain differential cell counts. Total leukocyte concentrations were determined by diluting blood 1/6 in 21~/~, acetic acid containing methylene blue and enumerating nucleated cells in a hemocytometer chamber. Spleens were aseptically removed and weighed. Spleen cell suspensions were prepared by pressing the tissue through a stainless steel grid with a 3 ml syringe plunger. Erythrocytes were removed by lysis in 0.14 M NH4C1. BM cells were flushed from femurs with 5 ml of Iscoves's Dulbecco's minimum Eagle medium (IDMEM) containing 25 mM HEPES, L-glutamine, antibiotics (Terry Fox Laboratories, Vancouver, B.C.) and 101'~, heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT) using a 25 gauge needle. Viable cell counts were determined by Trypan blue dye exclusion. In vitro colony assays To assess levels of myeloid progenitor cells (CFU-GM) in the BM and spleen, cells from individual mice were plated at 6.7 or 10 x 10S/ml (spleen) or 5 x 104/ml (BM) in duplicate or triplicate 10 x 35 mm petri dishes containing 20°~, FCS, 10 mg/ml bovine serum albumin (Boehringer Mannheim, Dorval, Quebec), 3~0 pokeweed mitogen mouse spleen cell serum-free conditioned media (PWM-SCCM, Terry Fox

205 Laboratories) as a source of growth factors which contained interleukin- 1~ (IL- 1~, 2.7 pg/ml), granulocyte-macrophage colony stimulating factor (GM-CSF, 75 pg/ml) and interleukin-3 (15 units/ml) in I D M E M and 0.3~o agar. Following 7 days at 37 °C in 5 ~o CO2, 95 ~o air in a humidified incubator, plates were examined under an inverted microscope and clusters >_-40 cells were scored as a colony (CFU-GM). In a separate series of experiments, BM and spleen cells obtained from naive mice were incubated with Photofrin ® (0.5 ng- 10 #g/ml) under conditions described above for the colony assay, except PWM-SCCM was excluded from the cultures.

FACS analysis Equal numbers of spleen cells from 3 control or 3 mice injected 7 days previously with Photofrin ® (25 mg/kg) were pooled and incubated with saturating concentrations of rat IgG monoclonal antibodies to mouse CD3 (pan-T cell, clone KT3, Tomonari, 1988), CD4 (T-helper cell, clone KT174, Serotec, Toronto, Ont.), CD8 (cytotoxic T cell, clone KT15, Tomonari and Lovering, 1988), B220 (B cell-restricted isoform of CD45, clone RA3-6B2, Coffman et al., 1982), interleukin-2 receptor (IL-2R, clone AMT 13, Osawa and Diamantstein, 1984), F4/80 (macrophage, Austyn and Gordon, 1981), or MOMA-2 (macrophage, Kraal et al., 1987). Rat monclonal antibody LR-1 (Hutchings etal., 1985), reactive against mouse splenic B cells was of the IgM isotype. Antibodies were incubated with l x 10 6 cells in PBS containing 2~o FCS and 0.03~o NaN3 for 45 minutes on ice. Cells were washed twice and incubated with FITClabeled goat anti-rat lgG (Fab')e fragment (Bio/Can Scientific Inc., Mississauga, Ont.) or FITC-labelled mouse anti-rat IgM monoclonal antibody (MARM-4, Serotec) for a further 45 minutes. Ceils were washed, fixed in 1~o p-formaldehyde in PBS and 5000 cells were analyzed on an EPICS C flow cytometer (Coulter Corporation Inc., Hialeah, FL) to determine fluorescence intensity. Background staining was

arbitrarily set to approximately 5 ~o for the antiIgG-FITC second antibody and 2~o for the anti-IgM-FITC second antibody. Specific staining was determined by subtraction of the background result from the percent staining result obtained for each monoclonal antibody.

Mitogenic responses Spleen cells (1 x 106/ml) were cultured with Con A or LPS (0-40/~g/ml, Sigma) for 72 h at 37 °C, 5 ~o CO2 in RPMI 1640 (Terry Fox Laboratories) containing 25 mM HEPES, L-glutamine, 5 x 10-SM 2-mercaptoethanol, 5~o FCS, 40/~M non-essential amino acids, 40/~M Na pyruvate, penicillin (80 U/ml), streptomycin (80 /~g/ml), gentamycin (2 #g/ml) and fungizone (40 ng/ml) in 96-well microtiter plates at 0.2 ml/well. Antibiotics and media supplements were from Gibco BRL (Burlington, Ont.). Proliferative responses were assayed by the addition of MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma) (Mosmann, 1983) for the final 2 h of culture. One hundred #1 of culture supernatant was removed and 150 ffl of acidified isopropanol was added to each well and thoroughly mixed using a multi-channel pipetter. Color intensity was measured on a densitometer (Dynatech Laboratories, Alexandria, VA) at a wavelength of 590 nm. Results are given as the mean absorbance of 6 replicates. In a separate series of experiments, in order to determine if BPD or Photofrin ® has mitogenic activity, naive mouse spleen cells were cultured for 72 h with concentration gradients (0.5 ng-10 /~g/ml) of each compound and proliferative responses were measured as described above.

Effects of Photofrin ® following immunosuppressive therapy 5-FU (Sigma) was dissolved at 10 mg/ml in warm PBS, filter sterilized and administered i.v. at 150 mg/kg/. One and three days later, mice received either 5~o dextrose or Photofrin ® (10 mg/kg) i.p. Blood leukocyte concentrations and spleen cellularity were measured in 3-4 mice per group up to 8 days following the 5-FU injection.

206 Ten untreated litter mates served as controls (Day 0 mice) for this experiment.

mg/kg) revealed that significant increases in total leukocytes and lymphocytes had occurred (Fig. 1). Blood hematocrit was also greater in these mice (control mean=43.6~o, SD=2.4; Photofrin e mean = 45.3~o, SD = 2.0), but this difference was not statistically significant. No analysis of blood from BPD-injected mice was performed

Statistical analysis Statistical comparisons between a treatment group and its appropriate control were performed using Student's t-test. A value of p < 0 . 0 5 was considered statistically significant.

Spleen and B M colony assays Analysis of hematopoietic activity of spleen cells by in vitro colony-forming assays, demonstrated that a significant elevation in relative levels of splenic C F U - G M occurred 48-72 h following the injection of either Photofrin ® dose and up to 96 h following the injection of the higher Photofrin ® dose (Fig. 2). Absolute numbers of splenic C F U - G M remained significantly elevated 7 days post-injection in the mice given either dose of Photofrin ®, at levels comparable to that observed at 72 h (Fig. 3). Relative numbers of BM C F U - G M were significantly elevated 24-48 h following either Photofrin e dose and as late as 168 h following the lower dose (Fig. 2). There were no significant changes in the number of BM

Results

Influence of Photofrin ® and BPD on spleen and blood parameters Administration (i.p.) of Photofrin ® at 10 and 25 mg/kg produced significant increases in relative spleen weight and spleen cell numbers in mice sampled 7 days later (Table I). Comparable responses were observed when Photofrin ® was given i.v., although the increase in relative spleen weight was not statistically significant at the lower dose. BPD (10 mg/kg) given i.v. did not significantly alter the relative spleen weight or spleen cell number. Analysis of peripheral blood 7 days after the administration of Photofrin ® (25 TABLE I

Mean relative spleen weights, total and relative spleen cell numbers of intact mice injected with solvent, B P D or Photofrin ® 7 days previoulsy. The standard error of the mean is given in parentheses. Treatment

Solvent BPD Solvent Photofrin Photofrin Solvent Photofrin Photofrin

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0.33 0.40 0.42 0.47 0.50 0.37 0.53 0.58

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1.98 (0.12) 2.28 (0.35) 2.17 (0.18) 3.03* (0.29) 3.07* (0.20) 2.09 (0.07) 2.93 ~ (0.25) 3.39 ~ (0.35)

(0.06) (0.05) (0.03) (0.04) (0.05) (0.01) + (0.05) ~ (0.03)

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or spleen C F U - G M in the first 96 h following BPD administration (data not shown). In vitro colony formation was not observed when naive spleen or BM cells were cultured with Photofrin ® in the absence of PWM-SCCM.

Spleen cell FACS analysis FACS analysis of spleen cells obtained 7 days following Photofrin ® (25 mg/kg) revealed no change in the total number of cells which expressed T cell-(CD3, CD4, CD8) or B cellspecific (B220) cell surface antigens (Fig. 4). The total number of spleen cells which stained with the macrophage-specific reagents MOMA-2 and F4/80 were also unchanged by Photofrin ® treatment. However, there was a large increase in the number of cells which were labelled with the monoclonal antibody LR-1 in the spleens of the Photofrin®-treated mice. Spleen cells expressing IL-2R occurred with a low frequency in both treatment groups.

of normal mice injected i.p. with 5~o dextrose ( , ) or Photofrin ® at 10 mg/kg ( 0 ) and 25 mg/kg (O). Data is o/ _ presented as the mean percentage (/o) + the standard error (SE) of the result obtained for solvent-injected mice sampled in parallel. Each data point represents the result for 6-16 mice. Mean number of colonies/plate for control mice ranged from 136-146 (BM) and 70-95 (spleen). Few (0-2) colonies were formed in the absence of spleen conditioned media.

× p<0.05, *p < 0.005.

Mitogenic responses Proliferative responses to a dose gradient of LPS by spleen cells prepared from mice 7 days following Photofrin ® (25 mg/kg) were comparable to that of the controls (Fig. 5). However, spleen cells obtained from Photofrin-treated mice were more responsive to Con A at 1.25 and 2.5/~g/ml than spleen cells of the controls (Fig. 5). Control mice gave a greater response to Con A at 5 /~g/ml. The proliferative response to Con A by spleen cells prepared from mice given BPD 7 days previously was identical to that obtained for the controls (data not shown). Culture of naive

208 spleen cells with Photofrin ® or B P D for 72 h did not elicit proliferative responses as measured by the MTT assay. Absorbance values were at background level at all porphyrin concentrations tested.

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CELL SURFACE ANTICEN Fig. 4. Expression of leukocyte surface antigens by spleen cells obtained from normal mice injected 7 days previously with 5° o dextrose (m) or Photofrin ® (25 mg/kg, D). Results are given as the mean + 1 SD of at least 3 separate FACS analyses. Values were obtained by multiplying total spleen cell number by the staining (% positive cells) result for each monoclonal antibody. *P < 0.01.

Discussion

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The ability of Photofrin ® to influence the immunohematopoietic system was suggested by earlier studies that showed it caused splenomegaly in normal mice (Gomer, 1988). A series of investigations by Canti et al. (1984, 1989) indicated that Photofrin ® and D H E could accelerate hematopoietic recovery of mice sub-lethally irradiated or treated with cytotoxic drugs. However, these previous studies did not examine mechanisms which might account for these responses or identify the cell lineages whose formation was affected by Photofrin ®. Evidence that Photofrin ® affects progenitor cells of the granulocyte-macrophage lineage was demonstrated in our studies by significant increases in C F U - G M in the BM and spleen 2-3 days following drug administration. This activation was sustained as total splenic C F U - G M remained significantly elevated 7 days later.

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ug/ml Fig. 5. Proliferative responses to LPS and Con A by spleen cells obtained from normal mice 7 days following i.p. injection of 5% dextrose ( 0 ) or Photofrin ® (25 mg/kg, © ) as determined by the MTT colormetric assay. Data points represent the mean + SD for three individual mice. Mean absorbance (A) values in the control wells (no mitogen) were 0.163 for the controls and 0.150 for Photofrin ®-treated mice. Four additional experiments with Con A and one with LPS gave similar results.

Splenic hypercellularity was not associated with an increase in the number of splenocytes which expressed T cell (CD3, CD4, CD8) or B cell (B220) markers. The total number of spleen cells which stained with monoclonal antibody LR-1 was approximately doubled 7 days following Photofrin ®. LR-1 is described as a mouse splenic B cell-specific reagent (Hutchings, 1985). However, in our hands there was an incomplete correlation with the degree of staining obtained with monoclonal antibodies specific for the B cell-restricted isoform of CD45 (B220) or/~ immunoglobulin chains (data not shown), in that the LR-1 reagent appears to overestimate numbers of splenic B cells. It has been observed that

Fig. 6. Mean + 1 SE blood leukocyte concentrations and nucleated spleen cell numbers in mice given 5?0 dextrose ( 0 ) or Photofrin ® (10 mg/kg, © ) on days 1 and 3, following treatment with 5-FU on day 0. Spleen data has been normalized to the number of cells per gram bodyweight. *p < 0.025.

LR-1 labels a high proportion of BM cells and that treatment of BM cells with LR-1 plus complement enriches for granulocytemacrophage progenitors (Marshall-Clarke, unpublished observations). Thus, the increased number of L R - I + cells in the spleens of Photofrin®-treated mice indicates that this antibody may recognize a differentiation antigen present on newly formed myeloid lineage cells in addition to mature splenic B cells. Administration of Photofrin ® did not appear to impair immunological responses of normal mice, since spleen cells from these mice retained strong mitogenic responses to LPS and Con A, although the mitogenic response to sub-optimal concentrations of Con A was greater in the Photofrin Otreated mice. The response to LPS, which is mitogenic for murine B cells, was similar for spleen cells prepared from control and

210 Photofrin®-treated mice. Photofrin ® caused a significant increase in total blood leukocyte and lymphocyte numbers. It is not clear whether the increase in blood lymphocytes is associated with increased production and/or a redistribution of lymphoid cells in these mice. That BPD does not modify the murine immunohematopoietic axis, indicates that hematopoietic stimulation is not a universal response following the administration of a porphyrin photosensitizer. The rapid in vivo clearance of BPD (Richter etal., 1990) may prevent this compound from influencing hematopoiesis. In contrast, Photofrin ® is detectable in various tissues for extended periods following its administration (Bellnier et al., 1989) and is known to distribute at quite high concentrations to both the spleen and BM. The mechanism by which Photofrin ® stimulates myelopoiesis in the mouse is presently not understood. In addition, since Photofrin ® is a complex mixture of porphyrin monomers, dimers and oligomers (Dougherty, 1987), the exact molecular identity of the hematostimulatory component is unknown and whether it corresponds to the photosensitising fraction. It is conceivable that Photofrin ® indirectly stimulates BM and spleen progenitor cells by eliciting the release of cytokines from hematopoietic accessory cells such as macrophages, fibroblasts, T lymphocytes or endothelial cells. It has been demonstrated that murine macrophages actively take up Photofrin ® (Korbelik et al., 1991). Macrophages produce a plethora of cytokines, constitutively or upon activation, which influence the activity of hematopoietic stem cells (Metcalf, 1991). However, Photofrin ® did not cause detectable changes in serum IL-I~, interleukin-6 or GMCSF (data not shown), factors produced by macrophages as well as other cell types, that influence the growth and differentiation of hematopoietic precursors (Metcalf, 1989, 1990). Accelerated hematopoietic recovery was observed in mice treated with Photofrin ® following myeloblation with 5-FU. Significant increases in blood leukocyte and spleen cell numbers were

observed 7-8 days later. These observations substantiate findings of an earlier study (Canti et al., 1989). Thus, the ability of the hematopoietic system to respond to Photofrin ® does not appear to be compromised by immunosuppressive therapy. Hemin (Stenzel et al., 1981) and metalloporphyrins but not protoporphyrin IX (Novogrodsky et al., 1989) are mitogenic for human T cells when cultured with macrophages as accessory cells. The mechanism by which these compounds induce mitogenesis has not been identified, but a perturbation of the cytoskeleton was proposed (Novogrodsky et al., 1989). It is unlikely Photoftin ® exerts effects on hematopoiesis through T cell activation. Proliferative responses were not observed when Photofrin ® was cultured with normal mouse spleen cells. In addition, total numbers of splenic CD3 + T cells and spleen cell expression of the T cell activation marker IL-2R (Osawa and Diamantstein, 1984) were unchanged by Photofrin ® administration. Activation of soluble guanylate cyclase by protoporphyrin IX has been demonstrated (Ignarro, 1989). Since Photofrin ® is structurally related to protoporhyrin IX, it is possible that it could activate hematopoietic progenitor cells by altering levels of the intracellular signal molecule cGMP, through modulation of guanylate cyclase activity. The influence of Photofrin ® on the activity of this enzyme in hematopoietic progenitor cells awaits clarification. It has been shown that c G M P increases C F U - G M by mouse BM cells in colony assays, possibly through an activation of resting stem cells (Oshita et al., 1977). Finally, it is conceivable that Photofrin ® affects hematopoiesis by entering the iron/heme metabolic pathway. It has been shown that Photofrin ® increases fibroblast synthesis of heme oxygenase, an enzyme involved in the degradation of heme (Gomer et al., 1991). Protoporphyrin IX increases expression of transferrin receptor and reduces intracellular levels of ferritin by human leukemic cell lines (Louache et al., 1984). Ferritin may act as a physiological regulator of granulopoiesis (Broxmeyer et al., 1978). A decrease in

211 the level of intracellular ferritin may permit enhanced growth and differentiation of myeloid progenitor cells. The hematostimulatory effects of Photofrin ® demonstrate that its in vivo properties are not restricted to that of a photosensitizer. Further analysis of the action of Photofrin ® and related compounds on the immunohematopoietic axis may provide insights into the role of porphyrins in the regulation of hematopoiesis.

Acknowledgements This work was supported, in part, by Research Grant 22070EC05 from the Defence Industrial Research Program (Ottawa, Canada) and a British Columbia Science Council G.R.E.A.T. Award to D.W.C.H. The authors gratefully acknowledge the technical assistance of Ms. Corina Dyck and Mr. Martin Renke.

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