British yournal of Dermatology (1982) 106, 687-695.

Pharmacological modulation of neutrophilphagocytic function in a patient with

recurrent sepsis, pyoderma gangrenosum andimpaired phagocytosis

C.B.BENTLEY-PHILLIPS, R.C.COOPER AND A.F.HALLETT

Departments of Pharmacology, Microbiology and Dermatology, University of Natal, South Africa

Accepted for publication 5 August 1981

SUMMARY

A 50-year-old man with recurrent life-threatening sepsis and a cutaneous condition resemblingpyoderma gangrenosum, was found to have a defect of neutrophil phagocytic function.Phagocytosis could be enhanced by corticosteroids, both in vitro in a dose-dependent manner,and in vivo, when it was accompanied by rapid clinical improvement. Studies with steroidhormones and immuno-stimulatory drugs are described.

In recent years various disorders of phagocytic function have been described. These disordersmay be due to impaired mobility, chemotaxis, ingestion, activation or microbicidal activity ineither the neutrophil polymorphonuclcar (PMN) leucocyte or the macrophage- Humoral orcellular immunity may also be abnormal, affecting the complement system or lymphokines,both of which are involved in phagocytic function.

Most of the recorded cases of neutrophil dysfunction have been due to impaired chemotaxis,often associated with other abnormalities of phagocytosis or killing defects. Certain microbici-dal disorders have also been ascribed to enzyme deficiencies. However, to our knowledge, anisolated defect in phagocytosis has not previously been described.

We describe a patient with such an isolated defect, who presented with recurrentlife-threatening sepsis and a dermatological picture resembling pyoderma gangrenosum. Thisdefect gave us the opportunity to study the effect upon it of certain pharmacological agents.Neutrophil phagocytosis may be enhanced by levamisole (Verhacgen, DeCock & De Cree, 1976;Ippen & Quadripur, 1975) and by clofazimine (Brandt, 1971). Pyoderma gangrenosum oftenresponds well to corticosteroids (Holt et al.^ 1980) and to clofazimine (Michaelsson et a/., 1976).

Correspondence: Dr C.B.Bcniley-Phillips, Department of Pharmacology, University of Natal, P.O. Box 17039,Congclla 4013, Natal, South Africa.

0007-0963/82/o6oo-o687$02.oo © 1982 British Association of Dermatologists

687

688 C.B.Bentley-Phillips, R.C.Cooper and A.F.HallettIn vitro and in vivo investigations were carried out with these drugs in an attempt to find a meansof influencing the patient's phagocytic defect.

CASE REPORT

The patient, a 50-year-old fitter and turner of Dutch extraction, presented in January 1978 witha 5-year history of intermittent fever, malaise, suppurative otitis media, abscesses andulceration. Twelve months previously, he had developed bronchopncumonia, pulmonaryabscesses and hidradenitis suppurativa of the axillae and inguinal regions. Excision of theaxillary vaults and grafting was carried out, but he developed post-operative septicaemia and asplenic abscess, which required splenectomy. He now had a 4-month-old progressing,destructive ulcer (12 x 15 cm) on his chest wall with irregular margins and overhanging edges(Fig. i). On his trunk and arms were numerous erythematous papules and pustules (Fig. 2)many of which became necrotic, ulcerated and pyodermatous. He also had a tonsillar abscessand appeared ill, but was apyrexial. A diagnosis of pyoderma gangrenosum was made, and animmune deficiency was suspected.

InvestigationsThe ESR was 112 mm/h (Westergren); Hb 120 g/100 ml; WBC 15,000/mm' (62*!o neutrophils,34",, lymphocytes). Urea, electrolytes, liver function tests and enzymes, and scrum cicctrophor-esis were normal, but there was mild carbohydrate intolerance. Rheumatoid factor, anti-nuclear

FIGURE 1. Rapidly progressive destructive ulcer with undermined edges on left chest wall.

Modulation of neutrophil phagocytic function 689

.,-««t

FIGURE 2. Early skin lesioti wiili IIDM rormation.

factor and auto-antibody screening were negative. Immunoglobulin and complement levelswere normal, Urinalysis was normal, and no Bence Jones protein was detected. The chest X-rayshowed old fibrotic change only. Sigmoidoscopy and barium enema were normal, showing noevidence of ulcerative colitis. Biopsies of the advancing edge of the ulcer and of new papularlesions showed oedematous and hyperplastic epidermis, acute ulceration with abscess formationand extensive mixed inflammatory infiltrate. Vascular changes were not marked. Directimmunofluorescence of early lesions was negative for deposits of IgG, IgA, IgM, Clq, C3, C4and fibrin. Cultures from the ulcers grew a variety of organisms but cultures from intactpustules were sterile. Circulating immune complexes were not detected. T and B lymphocyteswere present in normal numbers. Lymphocyte function, as assessed by PHA-inducedtransformation and migration inhibition, was normal. The rise in antibody titre afteradministration of tetanus toxoid indicated no abnormality of humoral immunity. The bonemarrow was normal. The neutrophil function tests are described below.

MATERIALS AND METHODS

Neutrophil function testsChemotaxis of PMN leucocytes was measured using a modification (Wilkinson, 1974) ofBoyden's technique (Boyden, 1962) and was estimated by measuring the number of PMNs thathad travelled through a millipore filter towards a chemo-attractant in a standard time. Thenitroblue tetrazolium (NBT) reduction test was carried cut on unstimulated PMNs, according

690 C.B.Bentley-Phillips, R.C.Cooper and A.F.Hallettto the method of Park et al. (1969), and also on PMNs stimulated with endotoxin (Park & Good,1970).

The phagocytic assay was carried out by incubating PMNs with Staphylococcus aureusorganisms. Leucocytes were obtained by sedimentation on a dextran-saline gradient; the purityof the neutrophil suspension thus produced was always in excess of 90",,. Tubes were set upwith (i) patient's cells-i-patient's serum + bacteria, (2) patient's cells + control plasma + bac-teria, and (3) control cells 4-patient's plasma-H bacteria. The percentage of phagocytosingPMNs and the average number of bacteria ingested per cell were determined. Tbe assay was alsocarried out using Candida albicans organisms or latex particles coated with IgG.

Intracellular killing was assessed by diluting the suspension of leucocytes and organisms withdistilled water, disrupting the cells and releasing the organisms. The surviving bacteria wereincubated on nutrient agar piatcs and the colonies were counted after 24 h. In order further todifferentiate defective phagocytosis from impaired intracellular killing, a modified assay ofneutrophil function using Lysostaphin (Tan ei al.y 1971) was also used. LyophilizedLysostaphin (Mead Johnson) was added to the final suspension before disruption of the PMNswith distilled water. This eliminated extracellular bacteria, but does not penetrate theneutrophil and it can be inactivated by trypsin before the lytic disruption of the PMNs.

TABLE I. Tests of phagocytic and baaericidal function

phagocytic indexPatient ceils + patient serumPatient cells + normal serumNormal cells + normal serumNormal cells + patient serum

Bactericidal assayPatient cells + patient serumNormal cells + normal serum (tst control)Normal cells + normal serum (2nd control)

Bactericidal as.(ay with lysostaphin f 25 ygjml)Patient cells \- patient scrumNormal cells H- normal serumPatient cells -t- patient serum + lysostaphinNormal cells -i- normal serum -1- lysostaphin

Percentage ofphagocytosing

neutrophils

0 min 120 min

15%

61%

Percentage ofsurviving bacteria

0 min 120 min

52 X 10" 35 X 10"

4 2 X 10* 1-5 X 10"

5 ox to* I-ox 10"

Percentageof surviving bacteria

0 min 120 min

48 X 10'' 3'9 X 10'43x10^ 8 9>«io''2'4x 10' 9-1 X 104-6 X 10' 46 X 10

Modulation of neutrophil phagocytic function 691

Pharmacological studiesThe effects of various pharmacological agents were assessed in vitro and in vivo. The in vitrostudies were performed by including the drug in the incubation mixture of leucocytes, serumand organisms and carrying out the phagocytic assay as described.

The drugs were incubated in serum for 30 min at 'ij'^C prior to assay3 in order to allow proteinbinding to stabilize. Where possible, a number of concentrations of the drug were used.However, in most cases it was only practical to use one concentration, and this was chosen to

TABLE 2. Studies showing effects on phagocytosis of in vitroadministration of drugs to suspension of neutrophils and

bacteria

In-vitro studies

Experiment iLevamisole (i x io""%Levamisole (i x io" ' ' \No drug addedNo drug added

Experiment 2Clofazimine (i x io"*iPrednisone (27 x io~Prednisone (2'7x 10"'Hydrocortisone (2-8 xNo drug addedNo drug added

Experiment 3Hydrocortisone (2'8 xTestosterone (29 x 10Nandrolone (2-5 x 10"Dexamethasone (i-o xFludrocoriisone (2-4 xNo drug addedNo drug added

Experiment 4No drug addedHydrocortisone (28 xHydrocortisone (2-8 xHydrocortisone (28 xHydrocortisone (2-8 xHydrocortisone (2-8 xHydrocortisone (28 xHydrocortisone (28 xHydrocortisone (28 xNo drug added

0

H)'M)

10 ^"M)

10 " ' ^ M )

IO"^M)10 "*M)

10-^M)10" ""M)

10 ""^M)

IO~*M)

10 " * M )

1 0 ' - ' M )

10-^M)

10 "•'M)

PC/PSNC/NSPC/PSNC/NS

PQPSPC/PSPC/PSPC/PSPC/PSNC/NS

PC/PSPC/PSPC/PSPC/PSPC/PSPC/PSNC/NS

PC/PSPC/PSPC/PSPC/PSPC/PSPC/PSPC/PSPC/PSNC/NSNC/NS

120 minPercentage ofphagocytosing

neutrophils

46%7 0 %2 6 %68%

4 2 %

54%64%7 0 %

2 4 %76%

7 1 %7 0 %66%

65%6 2 %2 8 %

66%

2 6 %

40%48%

53%6 0 %6 0 %

8 0 %

56%7 0 %

66",,

PC = Patient's cells; PS = patient's serum; NC = nornul con-trol's cells; NS = control's serutn.

692 C.B.Bentley-Phillips, R.C.Cooper and A.F.HallettTABLE 3. In vivo Studies showing the effects on phagocytosis

after administration of drugs

Prednisone 20 mg t.d.s.

Hydrocortisone 100 mg t.d.s.

Clofazimine 100 mg t.d.s.

Levamisole 150 mg daily

PC/PSNC/NSPC/PSNC/NSPC/PSPC/PSNC/NSPC/PSNC'NS

120 minPercentage ofphagocy cosing

neutrophils

56%74%7 0 %

7 2 %

48%45%7 0 %

55%66",,

approximate to levels of the drug usually achieved on administration of pharmacological dosesto human subjects, as derived from available pharmacokinetic data. The in vivo studies werecarried out after the patient had been on pharmacological doses of the drug for 96 h, the bloodbeing taken 2 h after the last dose. The drugs, concentrations and doses are shown in Tables 2and 3.

RESULTS

Neutrophil function testsThe resting and stimulated NBT reduction tests were within nonnal limits. Cytochemicalstudies of the neutrophil showed no enzyme defects (staining was carried out for myeloperoxi-dase, Sudan black, non-specific esterase, chloroacetate esterase, PAS, acid and alkalinephosphatase). Tests for chemotaxis were normal.

The patient's phagocytic activity was found to be markedly depressed (Table i), restorationbeing achieved by substitution of normal leucocytes but not normal serum. An intrinsic cellulardefect was therefore suspected associated with impaired killing in the bactericidal assay. Themarked reduction in surviving organisms in this assay after addition of Lysostaphin indicatesthat the organisms were extracellular, had not been phagocytosed and were thereforeunprotected from the lytic effects of this enzyme. The defect is therefore one of phagocytosisrather than of intracellular killing. This evidence that intracellular killing was normal wassupported by the normal NBT tests, the absence of cytochemical abnormalities, and normalpost-phagocytosis hexose monophosphate shunt activity and protein iodination. Phagocyticassays were also performed using Candida albicans and immunoglobulin-coated latex particles,and were found to be normal, suggesting that the phagocytic defect may be specific for certainorganisms only. Electron microscopy was performed on incubating leucocytes and organisms.The neutrophils appeared normal, but there was a major difference in the structure of theorganisms in the patient's cells compared with those in control neutrophils. The formerappeared to have retained their structure, the latter had not, suggesting the presence ofimpairment of bactericidal activity.

The in vitro studies (Table 2) show that phagocytosis was increased by levamisole.

Modulation of neutrophil phagocytic function 693clofazimine and various steroid hormones, but corticosteroids produced the most markedincrease, and this was demonstrable as a positive dose response. The in vivo studies (Table 3)again showed that phagocytosis was enhanced by corticosteroids, and to a lesser extent bylevamisole and clofazimine.

TREATMENT AND PROGRESS

Treatment with local measures and systemic antibiotics produced minimal improvement, but a3-weck course of prednisone (20 mg t.d.s., tailing off gradually) produced rapid healing of all thesmaller lesions. The ulcer on the chest wall also healed completely after 2 weeks, but 2 monthslater there developed rapidly progressive ulcers elsewhere on chest and abdomen.

A series of week-long courses of levamisole, hydrocortisone, clofazimine and prednisone,with a week's 'wash-out' period between each course, was administered, and phagocytic assayswere carried out during each course. Improvement of the smaller lesions and immediatecessation of symptoms of malaise were noticed with prednisone and hydrocortisone, but notwith clofazimine and levamisole. The corticosteroids restored phagocytic function to normal,whereas the assays were only moderately affected by the other drugs.

Over the next year, longer courses of prednisone were administered. It was found repeatedlythat clearing could be induced within 4 weeks, but remissions were short, and smallmaintenance doses were required. As marked Cushingoid features together with an appreciablegain in weight were developing it was decided to assess other steroid hormones. Phagocyticassays indicated that nandrolone and testosterone should be tried, and testosterone was found toproduce a partial improvement. However, it did not permit the withdrawal of corticosteroids,although a small sparing effect was achieved. Cytotoxic drugs have proved ineffective, and hehas had to continue with slowly increasing doses of prednisone for the past 2 years. Nohaematological or immunoglobulin changes have occurred.

DISCUSSION

An ideal model for the study of the clinical consequences of depressed phagocytosis and for theinvestigation of therapeutic agents designed to correct such a defect, would require a patientwith an immunological deficiency confined to phagocytic dysfunction. Extensive investigationin our patient has attempted to exclude defects in T and B lymphocyte function, disorders ofchemotaxis, abnormalities of circulating serum factors and intraceilular killing defects.Although mononuclear phagocytosis has not been studied, our findings indicate that thispatient's disorder is due to a pure defect of the phagocytic process.

The exact nature of this defect is unclear. Many of the recorded cases of neutrophildysfunction have been associated with severe systemic illness, such a neoplasia and malnutri-tion. The only systemic illness affecting this patient was pyoderma gangrenosum with recurrentsepsis, which may well be the result of the neutrophil disorder. It was repeatedly noted thatdrug-induced restoration of phagocytic activity was accompanied by marked clinical improve-ment. It is conceivable that the phagocytic dysfunction was secondary to widespread infection,but the rapid IH vitro correction of this dysfunction makes this unlikely. The good correlationbetween clinical effectiveness and the effects on phagocytosis suggests that the use of batteries ofdrugs in in vitro assays may help to assess therapeutic potential in cases of neutrophildysfunction.

It has been suggested that the splenectomy may have reduced the concentration of circulating

694 C.B.Bentley-Phillips, R.C.Cooper and A.F.Hallerttuftsin, deficiency of which leads to defective phagocytosis and increased susceptibility toinfections. However the recurrent infections predated the splcnectomy, which was performedbecause of a splenic abscess developing during a septicaemic episode. Also, investigationsshowed that the impaired phagocytosis was due to a cellular rather than a circulating serumfactor.

As the patient's chemotaxis was normal, it was felt that microfilament and microtubulefunction must be normal, and that the abnormality was more likely to be at surface receptor ortransmitter level. This was supported by the enhancing effect of the corticosteroids which,although they could be acting in vivo by reducing levels of anti-neutrophil antibodies, would notbe expected to have an effect in vitro. The fact that other steroid hormones with widely diversepharmacological actions all produced some restoration in in vitro phagocytic function, wouldsuggest some common structural grouping of the steroid molecule that is agonist at abnormalsurface receptors. The effects of corticosteroids on phagocytosis in this patient are surprising.Depression of phagocytic activity has been described in patients receiving high dose ofcorticosteroids (Greendyketv^/., 1965; Handin & Stossel, 1975), and very high doses of thesedrugs can inhibit normal phagocytosis in vitro (Stossel et al., 1972). However, a recent case ofpyoderma grangrenosum was described with impaired monocytic phagocytosis, as well as otherabnormalities of monocyte neutrophil chemotaxis. Addition of hydrocortisone in vitroenhanced phagocytosis (Norris et al., 1978).

Various immunological disorders have been described in pyoderma gangrenosum. Miller &Dooley (1973) reported a case with impaired random mobility and phagocytosis. In a series offifteen patients, Holt et al. (1980) report abnormalities of neutrophil function in nine cases.Phagocytosis was abnormal in one case, and there was no consistent abnormality in the others.They considered it unlikely that these abnormalities were causally related to the skin disease.Certainly the variety of lymphocyte, monocyte and neutrophil dysfunction associated withpyoderma gangrenosum described by these and other workers, would indicate that there is nosingle aetiological mechanism that can be incriminated on an immunological basis. Thebeneficial effects of corticosteroids in this condition have also never been satisfactorilyexplained. In our patient, it would seem that the hydrocortisone is acting not through anyimmunosuppressive mechanism, but rather through a restorative action on already com-promised immunity. It is hoped that further studies on immunological abnormalities and theeffects on them of pharmacological agents may help to elucidate some of the factors involved inthe aetiology and treatment of this ill-understood condition.

ACKNOWLEDGMENTS

We would like to thank Dr A.W.Segal of the Clinical Research Centre, Northwick Park, for hisadvice and guidance in the investigation of this patient, Mr J.Dorling of the same institution forinterpretation of electron micrographs and Dr R.Anderson of the Department of Immunology,University of Pretoria, for performing assays of post-phagocytic metabolic activity.

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